Beta-catenin interacts with MyoD and regulates its transcription activity.
ABSTRACT: Wnt regulation of muscle development is thought to be mediated by the beta-catenin-TCF/LEF-dependent canonical pathway. Here we demonstrate that beta-catenin, not TCF/LEF, is required for muscle differentiation. We showed that beta-catenin interacts directly with MyoD, a basic helix-loop-helix transcription factor essential for muscle differentiation and enhances its binding to E box elements and transcriptional activity. MyoD-mediated transactivation is inhibited in muscle cells when beta-catenin is deficient or the interaction between MyoD and beta-catenin is disrupted. These results demonstrate that beta-catenin is necessary for MyoD function, identifying MyoD as an effector in the Wnt canonical pathway.
Project description:Canonical Wnts promote myoblast differentiation; however, the role of ?-catenin in adult myogenesis has been contentious, and its mechanism(s) unclear. Using CRISPR-generated ?-catenin-null primary adult mouse myoblasts, we found that ?-catenin was essential for morphological differentiation and timely deployment of the myogenic gene program. Alignment, elongation and fusion were grossly impaired in null cells, and myogenic gene expression was not coordinated with cytoskeletal and membrane remodeling events. Rescue studies and genome-wide analyses extended previous findings that a ?-catenin-TCF/LEF interaction is not required for differentiation, and that ?-catenin enhances MyoD binding to myogenic loci. We mapped cellular pathways controlled by ?-catenin and defined novel targets in myoblasts, including the fusogenic genes myomaker and myomixer. We also showed that interaction of ?-catenin with ?-catenin was important for efficient differentiation. Overall the study suggests dual roles for ?-catenin: a TCF/LEF-independent nuclear function that coordinates an extensive network of myogenic genes in cooperation with MyoD; and an ?-catenin-dependent membrane function that helps control cell-cell interactions. ?-Catenin-TCF/LEF complexes may function primarily in feedback regulation to control levels of ?-catenin and thus prevent precocious/excessive myoblast fusion.
Project description:Wnt3a activates the ;canonical' signaling pathway, stimulating the nuclear accumulation of beta-catenin and activation of Lef/Tcf-sensitive transcription of developmentally important genes. Using totipotent mouse F9 teratocarcinoma cells expressing frizzled-1 (Fz1), we investigated roles of tyrosine kinase activity in Wnt/beta-catenin signaling. Treatment with either genistein or Src family kinase inhibitor PP2 attenuates Wnt3a-stimulated Lef/Tcf transcription activation and primitive endoderm formation. siRNA-induced knockdown of Src likewise attenuates Lef/Tcf transcription and primitive endoderm formation in response to Wnt3a, implicating Src as a positive regulator of Wnt/beta-catenin signaling. We discovered that Src binds dishevelled-2 (Dvl2), a key phosphoprotein in Wnt signaling, at two positions: an SH3-binding domain and a C-terminal domain. The Y18F mutant of Dvl2 attenuates the Wnt3a-stimulated Lef/Tcf-sensitive transcriptional response. Wnt3a stimulates Src docking to Dvl2 and activation of this tyrosine kinase. Activated Src, in turn, enhances Wnt activation of the canonical pathway. We show that Dvl2 and beta-catenin are crucially important substrates for tyrosine phosphorylation in the canonical Wnt/beta-catenin pathway.
Project description:Beta-catenin is a key mediator in the canonical Wnt signaling pathway, which plays important roles in multiple developmental processes. Inappropriate activation of this pathway leads to developmental defects and development of certain cancers. Upon Wnt signaling, beta-catenin binds TCF/LEF transcription factors. The TCF/LEF-beta-catenin complex then recruits a variety of transcriptional coactivators to the promoter/enhancer region of Wnt-responsive genes and activates target gene transcription. In this article, we demonstrate that GRIP1-associated coactivator 63 (GAC63), a recently identified nuclear receptor (NR) coactivator, interacts with beta-catenin. The N-terminus of GAC63 is the binding site for beta-catenin, whereas a C-terminal fragment of beta-catenin including armadillo repeats 10-12 binds to GAC63. Over-expression of GAC63 enhanced the transcriptional activity of beta-catenin, and also greatly enhanced TCF/LEF-regulated reporter gene activity in a beta-catenin-dependent manner. Endogenous GAC63 was recruited to TCF/LEF-responsive enhancer elements when beta-catenin levels were induced by LiCl. In addition, reduction of endogenous GAC63 level by small interfering RNA (siRNA) inhibited TCF/LEF-mediated gene transcription. Our findings reveal a new function of GAC63 in transcriptional activation of Wnt-responsive genes.
Project description:During canonical Wnt signaling, the activity of nuclear β-catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view, we used the CRISPR/Cas9 genome editing approach to generate HEK 293T cell clones lacking all four TCF/LEF genes. By performing unbiased whole transcriptome sequencing analysis, we found that a subset of β-catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome-wide analysis that β-catenin occupied specific genomic regions in the absence of TCF/LEF. Finally, we revealed the existence of a transcriptional activity of β-catenin that specifically appears when TCF/LEF factors are absent, and refer to this as β-catenin-GHOST response. Collectively, this study uncovers a previously neglected modus operandi of β-catenin that bypasses the TCF/LEF transcription factors.
Project description:A major outcome of the canonical Wnt/beta-catenin-signalling pathway is the transcriptional activation of a specific set of target genes. A typical feature of the transcriptional response induced by Wnt signalling is the involvement of Tcf/Lef factors that function in the nucleus as the principal mediators of signalling. Vertebrate Tcf/Lef proteins perform two well-characterized functions: in association with beta-catenin they activate gene expression, and in the absence of Wnt ligands they bind TLE/Groucho proteins to act as transcriptional repressors. Although the general characteristics of Tcf/Lef factors are well understood, the mechanisms that control their specific roles in various cellular backgrounds are much less defined. In this report we reveal that the evolutionary conserved Dazap2 protein functions as a TCF-4 interacting partner. We demonstrate that a short region proximal to the TCF-4 HMG box mediates the interaction and that all Tcf/Lef family members associate with Dazap2. Interestingly, knockdown of Dazap2 not only reduced the activity of Wnt signalling as measured by Tcf/beta-catenin reporters but additionally altered the expression of Wnt-signalling target genes. Finally, chromatin immunoprecipitation studies indicate that Dazap2 modulates the affinity of TCF-4 for its DNA-recognition motif.
Project description:Atoh1, a basic helix-loop-helix transcription factor, plays a critical role in the differentiation of several epithelial and neural cell types. We found that beta-catenin, the key mediator of the canonical Wnt pathway, increased expression of Atoh1 in mouse neuroblastoma cells and neural progenitor cells, and baseline Atoh1 expression was decreased by siRNA directed at beta-catenin. The up-regulation of Atoh1 was caused by an interaction of beta-catenin with the Atoh1 enhancer that could be demonstrated by chromatin immunoprecipitation. We found that two putative Tcf-Lef sites in the 3' enhancer of the Atoh1 gene displayed an affinity for beta-catenin and were critical for the activation of Atoh1 transcription because mutation of either site decreased expression of a reporter gene downstream of the enhancer. Tcf-Lef co-activators were found in the complex that bound to these sites in the DNA together with beta-catenin. Inhibition of Notch signaling, which has previously been shown to induce bHLH transcription factor expression, increased beta-catenin expression in progenitor cells of the nervous system. Because this could be a mechanism for up-regulation of Atoh1 after inhibition of Notch, we tested whether siRNA to beta-catenin prevented the increase in Atoh1 and found that beta-catenin expression was required for increased expression of Atoh1 after Notch inhibition.
Project description:The canonical Wnt signaling pathway is critical for myogenesis and can induce muscle progenitors to switch from proliferation to differentiation; how Wnt signals integrate with muscle specific regulatory factors in this process is poorly understood. We previously demonstrated that the Barx2 homeobox protein promotes differentiation in cooperation with the muscle regulatory factor (MRF) MyoD. Pax7, another important muscle homeobox factor represses differentiation. We now identify Barx2,MyoD,and Pax7 as novel components of the Wnt effector complex, providing a new molecular pathway for regulation of muscle progenitor differentiation. Canonical Wnt signaling induces Barx2 expression in muscle progenitors and perturbation of Barx2 leads to misregulation of Wnt target genes. Barx2 activates two endogenous Wnt target promoters as well as the Wnt reporter gene TOPflash, the latter synergistically with MyoD. Moreover, Barx2 interacts with the core Wnt effectors β-catenin and TCF, is recruited to TCF/LEF sites, and promotes recruitment of β-catenin. In contrast, Pax7 represses the Wnt reporter gene and antagonizes the activating effect of Barx2. Pax7 also binds β-catenin suggesting that Barx2 and Pax7 may compete for interaction with the core Wnt effector complex. Overall, the data show for the first time that Barx2, Pax7, and MRFs can act as direct transcriptional effectors of Wnt signals in myoblasts and that Barx2 and Wnt signaling participate in a regulatory loop. We propose that antagonism between Barx2 and Pax7 in regulation of Wnt signaling may help mediate the switch from myoblast proliferation to differentiation. RNA-Seq analyses was used to characterize gene expression in primary myoblasts from wild-type and Barx2 knockout mice.
Project description:Beta-catenin plays a dual role as an adhesion molecule in adherens junctions at the plasma membrane and as a key intermediate in the canonical Wnt signalling pathway. The cytosolic soluble pool of beta-catenin, involved in the transmission of the Wnt signal, is normally subjected to rapid protein degradation. On activation of the Wnt cascade, beta-catenin becomes stabilized and then translocates into the nucleus where it co-activates transcription factors of the TCF (T-cell factor)/LEF (lymphoid enhancer factor) family. The expression of plasma membrane-targeted forms of beta-catenin has been shown to also activate TCF/LEF-dependent transcription and different mechanisms have been put forward. In the present study, we have undertaken a systematic analysis of the signalling capability of non-degradable forms of beta-catenin targeted to different cellular compartments. beta-Catenin targeted to the plasma membrane activated transcription to a greater extent compared with non-targeted beta-catenin, and led to a marked stabilization of cytosolic soluble beta-catenin. These effects were independent of the competition with endogenous beta-catenin for binding to E-cadherin at the plasma membrane, since targeting non-degradable beta-catenin to other cellular compartments, i.e. the outer mitochondrial membrane and the endoplasmic reticulum membrane, also resulted in the accumulation of cytosolic wild-type beta-catenin and activation of beta-catenin-dependent signalling. In contrast, nuclear-targeted beta-catenin was without significant effect on cytosolic wild-type beta-catenin and did not activate transcription. Our results suggest that cytosolic accumulation of beta-catenin is a prerequisite for the activation of TCF/LEF-dependent transcription in the nucleus.
Project description:Wnt signals control decisive steps in development and can induce the formation of tumors. Canonical Wnt signals control the formation of the embryonic axis, and are mediated by stabilization and interaction of beta-catenin with Lef/Tcf transcription factors. An alternative branch of the Wnt pathway uses JNK to establish planar cell polarity in Drosophila and gastrulation movements in vertebrates. We describe here the vertebrate protein Diversin that interacts with two components of the canonical Wnt pathway, Casein kinase Iepsilon (CKIepsilon) and Axin/Conductin. Diversin recruits CKIepsilon to the beta-catenin degradation complex that consists of Axin/Conductin and GSK3beta and allows efficient phosphorylation of beta-catenin, thereby inhibiting beta-catenin/Tcf signals. Morpholino-based gene ablation in zebrafish shows that Diversin is crucial for axis formation, which depends on beta-catenin signaling. Diversin is also involved in JNK activation and gastrulation movements in zebrafish. Diversin is distantly related to Diego of Drosophila, which functions only in the pathway that controls planar cell polarity. Our data show that Diversin is an essential component of the Wnt-signaling pathway and acts as a molecular switch, which suppresses Wnt signals mediated by the canonical beta-catenin pathway and stimulates signaling via JNK.
Project description:During canonical Wnt signalling the activity of nuclear beta-catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view we used the CRISPR/Cas9 genome editing approach to generate HEK 293T cell clones simultaneously carrying loss-of-function alleles of all four TCF/LEF genes. Exploiting unbiased whole transcriptome sequencing studies, we found that a subset of beta-catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome-wide analysis that beta-catenin occupied specific genomic regions in the absence of TCF/LEF. Finally, we revealed the existence of a transcriptional activity of beta-catenin that specifically appears when TCF/LEF factors are absent, and refer to this as beta-catenin-GHOST response. Collectively, this study uncovers a previously neglected modus operandi of beta-catenin that bypasses the TCF/LEF transcription factors.