ABSTRACT: ?-Catenin (?-cat) is an actin-binding protein required for cell-cell cohesion. Although this adhesive function for ?-cat is well appreciated, cells contain a substantial amount of nonjunctional ?-cat that may be used for other functions. We show that ?-cat is a nuclear protein that can interact with ?-catenin (?-cat) and T-cell factor (TCF) and that the nuclear accumulation of ?-cat depends on ?-cat. Using overexpression, knockdown, and chromatin immunoprecipitation approaches, we show that ?-cat attenuates Wnt/?-cat-responsive genes in a manner that is downstream of ?-cat/TCF loading on promoters. Both ?-cat- and actin-binding domains of ?-cat are required to inhibit Wnt signaling. A nuclear-targeted form of ?-cat induces the formation of nuclear filamentous actin, whereas cells lacking ?-cat show altered nuclear actin properties. Formation of nuclear actin filaments correlates with reduced RNA synthesis and altered chromatin organization. Conversely, nuclear extracts made from cells lacking ?-cat show enhanced general transcription in vitro, an activity that can be partially rescued by restoring the C-terminal actin-binding region of ?-cat. These data demonstrate that ?-cat may limit gene expression by affecting nuclear actin organization.
Project description:Aberrant MYC gene expression by the Wnt/beta-catenin pathway is implicated in colorectal carcinogenesis. Wnt/beta-catenin signaling stimulates association of the beta-catenin coactivator complex with two Wnt responsive enhancers (WREs) located in close proximity to MYC gene boundaries. Each enhancer directly binds members of the TCF/Lef family of transcription factors that, in turn, recruit beta-catenin. In a previous report, we showed that the downstream MYC enhancer (MYC 3' WRE) cooperated with the upstream enhancer (MYC 5' WRE) to activate expression of a heterologous reporter gene in response to Wnt/beta-catenin and mitogen signaling. Here we use chromatin conformation capture (3C) to show that the MYC 5' and 3' WREs are juxtaposed at the genomic MYC locus during active transcription. This MYC 5'3' chromatin loop is present in HCT116 human colorectal cancer cells that contain high levels of nuclear beta-catenin and is absent in HEK293 cells that contain trace amounts of nuclear beta-catenin. Depletion of functional beta-catenin/TCF complexes blocks formation of the MYC 5'3 chromatin loop. Furthermore, we find that the chromatin loop is absent in quiescent cells, but is rapidly and transiently induced by serum mitogens in a beta-catenin-dependent manner. Thus, we propose that a distinct chromatin architecture coordinated by beta-catenin/TCF-bound WREs accompanies transcriptional activation of MYC gene expression.
Project description:Wnt/?-catenin signaling elicits context-dependent transcription switches that determine normal development and oncogenesis. These are mediated by the Wnt enhanceosome, a multiprotein complex binding to the Pygo chromatin reader and acting through TCF/LEF-responsive enhancers. Pygo renders this complex Wnt-responsive, by capturing ?-catenin via the Legless/BCL9 adaptor. We used CRISPR/Cas9 genome engineering of Drosophila legless (lgs) and human BCL9 and B9L to show that the C-terminus downstream of their adaptor elements is crucial for Wnt responses. BioID proximity labeling revealed that BCL9 and B9L, like PYGO2, are constitutive components of the Wnt enhanceosome. Wnt-dependent docking of ?-catenin to the enhanceosome apparently causes a rearrangement that apposes the BCL9/B9L C-terminus to TCF. This C-terminus binds to the Groucho/TLE co-repressor, and also to the Chip/LDB1-SSDP enhanceosome core complex via an evolutionary conserved element. An unexpected link between BCL9/B9L, PYGO2 and nuclear co-receptor complexes suggests that these ?-catenin co-factors may coordinate Wnt and nuclear hormone responses.
Project description:Canonical Wnt signaling and its nuclear effectors, beta-catenin and the family of T-cell factor (TCF) DNA-binding proteins, belong to the small number of regulatory systems which are repeatedly used for context-dependent control of distinct genetic programs. The apparent ability to elicit a large variety of transcriptional responses necessitates that beta-catenin and TCFs distinguish precisely between genes to be activated and genes to remain silent in a specific context. How this is achieved is unclear. Here, we examined patterns of Wnt target gene activation and promoter occupancy by TCFs in different mouse cell culture models. Remarkably, within a given cell type only Wnt-responsive promoters are bound by specific subsets of TCFs, whereas nonresponsive Wnt target promoters remain unoccupied. Wnt-responsive, TCF-bound states correlate with DNA hypomethylation, histone H3 hyperacetylation, and H3K4 trimethylation. Inactive, nonresponsive promoter chromatin shows DNA hypermethylation, is devoid of active histone marks, and additionally can show repressive H3K27 trimethylation. Furthermore, chromatin structural states appear to be independent of Wnt pathway activity. Apparently, cell-type-specific regulation of Wnt target genes comprises multilayered control systems. These involve epigenetic modifications of promoter chromatin and differential promoter occupancy by functionally distinct TCF proteins, which together determine susceptibility to Wnt signaling.
Project description:Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder caused by a mutation of lamin A, which contributes to nuclear architecture and the spatial organization of chromatin in the nucleus. The expression of a lamin A mutant, named progerin, leads to functional and structural disruption of nuclear organization. Since progerin lacks a part of the actin-binding site of lamin A, we hypothesized that nuclear actin dynamics and function are altered in HGPS cells. Nuclear F-actin is required for the organization of nuclear shape, transcriptional regulation, DNA damage repair, and activation of Wnt/?-catenin signaling. Here we show that the expression of progerin decreases nuclear F-actin and impairs F-actin-regulated transcription. When nuclear F-actin levels are increased by overexpression of nuclear-targeted actin or by using jasplakinolide, a compound that stabilizes F-actin, the irregularity of nuclear shape and defects in gene expression can be reversed. These observations provide evidence for a novel relationship between nuclear actin and the etiology of HGPS.
Project description:BACKGROUND: beta-catenin is a key mediator of the canonical Wnt pathway as it associates with members of the T-cell factor (TCF) family at Wnt-responsive promoters to drive the transcription of Wnt target genes. Recently, we showed that Rac1 GTPase synergizes with beta-catenin to increase the activity of a TCF-responsive reporter. This synergy was dependent on the nuclear presence of Rac1, since inhibition of its nuclear localization effectively abolished the stimulatory effect of Rac1 on TCF-responsive reporter activity. We hypothesised that Rac1 plays a direct role in enhancing the transcription of endogenous Wnt target genes by modulating the beta-catenin/TCF transcription factor complex. RESULTS: We employed chromatin immunoprecipitation studies to demonstrate that Rac1 associates with the beta-catenin/TCF complex at Wnt-responsive promoters of target genes. This association served to facilitate transcription, since overexpression of active Rac1 augmented Wnt target gene activation, whereas depletion of endogenous Rac1 by RNA interference abrogated this effect. In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway. Tiam1 promoted the formation of a complex containing Rac1 and beta-catenin. Furthermore, endogenous Tiam1 associated with endogenous beta-catenin, and this interaction was enhanced in response to Wnt3a stimulation. Intriguingly, Tiam1 was recruited to Wnt-responsive promoters upon Wnt3a stimulation, whereas Rac1 was tethered to TCF binding elements in a Wnt-independent manner. CONCLUSION: Taken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active beta-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription. Our results demonstrate a novel functional mechanism underlying the cross-talk between Rac1 and the canonical Wnt signalling pathway.
Project description:Dysregulation of canonical Wnt signaling is thought to play a role in colon carcinogenesis. ?-Catenin, a key mediator of the pathway, is stabilized upon Wnt activation and accumulates in the nucleus, where it can interact with the transcription factor T cell factor (TCF) to transactivate gene expression. Normal colonic epithelia express a truncated TCF-1 form, called dnTCF-1, that lacks the critical ?-catenin-binding domain and behaves as a transcriptional suppressor. How the cell maintains a balance between the two forms of TCF-1 is unclear. Here, we show that ERM-binding phosphoprotein 50 (EBP50) modulates the interaction between ?-catenin and TCF-1. We observed EBP50 localization to the nucleus of human colorectal carcinoma cell lines at low cell culture densities and human primary colorectal tumors that manifested a poor clinical outcome. In contrast, EBP50 was primarily membranous in confluent cell lines. Aberrantly located EBP50 stabilized conventional ?-catenin/TCF-1 complexes and connected ?-catenin to dnTCF-1 to form a ternary molecular complex that enhanced Wnt/?-catenin signaling events, including the transcription of downstream oncogenes such as c-Myc and cyclin D1. Genome-wide analysis of the EBP50 occupancy pattern revealed consensus binding motifs bearing similarity to Wnt-responsive element. Conventional chromatin immunoprecipitation assays confirmed that EBP50 bound to genomic regions highly enriched with TCF/LEF binding motifs. Knockdown of EBP50 in human colorectal carcinoma cell lines compromised cell cycle progression, anchorage-independent growth, and tumorigenesis in nude mice. We therefore suggest that nuclear EBP50 facilitates colon tumorigenesis by modulating the interaction between ?-catenin and TCF-1.
Project description:In canonical Wnt signaling, Dishevelled (Dvl) is a critical cytoplasmic regulator that releases beta-catenin from degradation. Here, we find that Dvl and c-Jun form a complex with beta-catenin-T-cell factor 4 (TCF-4) on the promoter of Wnt target genes and regulate gene transcription. The complex forms via two interactions of nuclear Dvl with c-Jun and beta-catenin, respectively, both of which bind to TCF. Disrupting the interaction of Dvl with either c-Jun or beta-catenin suppresses canonical Wnt signaling-stimulated transcription, and the reduction of Dvl diminished beta-catenin-TCF-4 association on Wnt target gene promoters in vivo. Expression of a TCF-Dvl fusion protein largely rescued the c-Jun knockdown Wnt signaling deficiency in mammalian cells and zebrafish. Thus, we confirm that c-Jun functions in canonical Wnt signaling and show that c-Jun functions as a scaffold in the beta-catenin-TCFs transcription complex bridging Dvl to TCF. Our results reveal a mechanism by which nuclear Dvl cooperates with c-Jun to regulate gene transcription stimulated by the canonical Wnt signaling pathway.
Project description:Recent studies suggest the importance of the transition of airway epithelial cells (EMT) in pulmonary fibrosis, and also indicate a role for Wingless protein (Wnt)/?-catenin signaling in idiopathic pulmonary fibrosis. We investigated the possible role of the Wnt signaling pathway in inducing EMT in lung epithelial cells, and sought to unravel the role of c-Jun-N-terminal-kinase-1 (JNK1). The exposure of C10 lung epithelial cells or primary mouse tracheal epithelial cells (MTECs) to Wnt3a resulted in increases in JNK phosphorylation and nuclear ?-catenin content. Because the role of ?-catenin as a transcriptional coactivator is well established, we investigated T-cell factor/lymphocyte-enhancement factor (TCF/LEF) transcriptional activity in C10 lung epithelial cells after the activation of Wnt. TCF/LEF transcriptional activity was enhanced after the activation of Wnt, and this increase in TCF/LEF transcriptional activity was diminished after the small interfering (si)RNA-mediated ablation of JNK. The activation of the Wnt pathway by Wnt3a, or the expression of either wild-type or constitutively active ?-catenin (S37A), led to the activation of an EMT transcriptome, manifested by the increased mRNA expression of CArG box-binding factor-A, fibroblast-specific protein (FSP)-1, ?-smooth muscle actin (?-SMA), and vimentin, increases in the content of ?-SMA and FSP1, and the concomitant loss of zona occludens-1. The siRNA-mediated ablation of ?-catenin substantially decreased Wnt3a-induced EMT. The siRNA ablation of JNK1 largely abolished Wnt3a, ?-catenin, and ?-catenin S37a-induced EMT. In MTECs lacking Jnk1, Wnt3a-induced increases in nuclear ?-catenin, EMT transcriptome, and the content of ?-SMA or FSP1 were substantially diminished. These data show that the activation of the Wnt signaling pathway is capable of inducing an EMT program in lung epithelial cells through ?-catenin, and that this process is controlled by JNK1.
Project description:The ?-catenin transcriptional coactivator is the key mediator of the canonical Wnt signaling pathway. In the absence of Wnt, ?-catenin associates with a cytosolic and multi-protein destruction complex where it is phosphorylated and targeted for proteasomal degradation. In the presence of Wnt, the destruction complex is inactivated and ?-catenin translocates into the nucleus. In the nucleus, ?-catenin binds T-cell factor (TCF) transcription factors to activate expression of c-MYC (MYC) and Axis inhibition protein 2 (AXIN2). AXIN2 is a member of the destruction complex and, thus, serves in a negative feedback loop to control Wnt/?-catenin signaling. AXIN2 is also present in the nucleus, but its function within this compartment is unknown. Here, we demonstrate that AXIN2 localizes to the nuclei of epithelial cells within normal and colonic tumor tissues as well as colorectal cancer cell lines. In the nucleus, AXIN2 represses expression of Wnt/?-catenin-responsive luciferase reporters and forms a complex with ?-catenin and TCF. We demonstrate that AXIN2 co-occupies ?-catenin/TCF complexes at the MYC promoter region. When constitutively localized to the nucleus, AXIN2 alters the chromatin structure at the MYC promoter and directly represses MYC gene expression. These findings suggest that nuclear AXIN2 functions as a rheostat to control MYC expression in response to Wnt/?-catenin signaling.
Project description:Osteoblasts exhibit complex Wnt-induced effects that increase T cell factor (TCF)/lymphoid enhancing factor-dependent transcription in parallel with beta-catenin stabilization and nuclear factor binding to TCF response element DNA. Here we show that Wnt-dependent gene expression increases during the early phase of osteoblast differentiation in vitro, is enhanced by prostaglandin E(2) activation of transcription factor Runx2 (runt homology domain transcription factor 2), and is specifically suppressed in Runx2 antisense-depleted osteoblasts. Moreover, Wnt pathway induction increases expression of the Runx2-sensitive gene, TGF-beta type I receptor, without increasing nuclear Runx2 levels or Runx2 binding to DNA. Rather, despite an increase in beta-catenin levels, Wnt pathway induction enhances Runx2 transcriptional potential in a beta-catenin-independent way. Runx2 functionally associates with TCF-4 that lacks a beta-catenin-binding domain and is more fully activated in response to both prostaglandin E(2) and Wnt pathway induction. Wnt pathway induction increases TGF-beta type I receptor expression, yet regulates, both positively and negatively, TGF-beta signaling. Furthermore, TGF-beta signaling enhances TCF-4 and lymphoid enhancing factor-1 mRNA expression and increases TCF-4 transcriptional activity. Therefore, we propose that cross talk between the Wnt and TGF-beta pathways, which converge on Runx2, both promotes and attenuates individual aspects of osteoblast maturation.