RA Acts in a Coherent Feed-Forward Mechanism with Tbx5 to Control Limb Bud Induction and Initiation.
ABSTRACT: The retinoic acid (RA)- and ?-catenin-signaling pathways regulate limb bud induction and initiation; however, their mechanisms of action are not understood and have been disputed. We demonstrate that both pathways are essential and that RA and ?-catenin/TCF/LEF signaling act cooperatively with Hox gene inputs to directly regulate Tbx5 expression. Furthermore, in contrast to previous models, we show that Tbx5 and Tbx4 expression in forelimb and hindlimb, respectively, are not sufficient for limb outgrowth and that input from RA is required. Collectively, our data indicate that RA signaling and Tbx genes act in a coherent feed-forward loop to regulate Fgf10 expression and, as a result, establish a positive feedback loop of FGF signaling between the limb mesenchyme and ectoderm. Our results incorporate RA-, ?-catenin/TCF/LEF-, and FGF-signaling pathways into a regulatory network acting to recruit cells of the embryo flank to become limb precursors.
Project description:Mouse F9 cells differentiate into primitive extraembryonic endoderm (PrE) when treated with retinoic acid (RA), and this is accompanied by an up-regulation of Gata6. The role of the GATA6 network in PrE differentiation is known, and we have shown it directly activates Wnt6. Canonical Wnt/?-catenin signaling is required by F9 cells to differentiate to PrE, and this, like most developmental processes, requires input from one or more additional pathways. We found both RA and Gata6 overexpression, can induce the expression of Indian Hedgehog (Ihh) and a subset of its target genes through Gli activation during PrE induction. Chemical activation of the Hh pathway using a Smoothened agonist (SAG) also increased Gli reporter activity, and as expected, when Hh signaling was blocked with a Smoothened antagonist, cyclopamine, this RA-induced reporter activity was reduced. Interestingly, SAG alone failed to induce markers of PrE differentiation, and had no effect on Wnt/?-catenin-dependent TCF-LEF reporter activity. The expected increase in Wnt/?-catenin-dependent TCF-LEF reporter activity and PrE markers induced by RA was, however, blocked by cyclopamine. Finally, inhibiting GSK3 activity with BIO increased both TCF-LEF and Gli reporter activities. Together, we demonstrate the involvement of Hh signaling in the RA-induced differentiation of F9 cells into PrE, and while the activation of the Hh pathway itself is not sufficient, it as well as active Wnt/?-catenin are necessary for F9 cell differentiation.
Project description:BackgroundThe Wnt/?-catenin signaling pathway is a prolific regulator of cell-to-cell communication and gene expression. Canonical Wnt/?-catenin signaling involves partnering of ?-catenin with members of the TCF/LEF family of transcription factors (TCF1, TCF3, TCF4, LEF1) to regulate gene expression. IL-6 is a key cytokine involved in inflammation and is particularly a hallmark of inflammation in the brain. Astrocytes, specialized glial cells in the brain, secrete IL-6. How astrocytes regulate IL-6 expression is not entirely clear, although in other cells NF?B and C/EBP pathways play a role. We evaluated here the interface between ?-catenin, TCFs/LEF and C/EBP and NF-?B in relation to IL-6 gene regulation in astrocytes.MethodsWe performed molecular loss and/or gain of function studies of ?-catenin, TCF/LEF, NF?B, and C/EBP to assess IL-6 regulation in human astrocytes. Specifically, siRNA mediated target gene knockdown, cDNA over expression of target gene, and pharmacological agents for regulation of target proteins were used. IL-6 levels was evaluated by real time quantitative PCR and ELISA. We also cloned the IL-6 promoter under a firefly luciferase reporter and used bioinformatics, site directed mutagenesis, and chromatin immunoprecipitation to probe the interaction between ?-catenin/TCFs/LEFs and IL-6 promoter activity.Results?-catenin binds to TCF/LEF to inhibits IL-6 while TCFs/LEF induce IL-6 transcription through interaction with ATF-2/SMADs. ?-catenin independent of TCFs/LEF positively regulates C/EBP and NF-?B, which in turn activate IL-6 expression. The IL-6 promoter has two putative regions for TCFs/LEF binding, a proximal site located at -91?nt and a distal site at -948?nt from the transcription start site, both required for TCF/LEF induction of IL-6 independent of ?-catenin.ConclusionIL-6 regulation in human astrocytes engages a discordant interaction between ?-catenin and TCF/LEF. These findings are intriguing given that no role for ?-catenin nor TCFs/LEF to date is associated with IL-6 regulation and suggest that ?-catenin expression in astrocytes is a critical regulator of anti-inflammatory responses and its disruption can potentially mediate persistent neuroinflammation. Video Abstract Graphical abstract
Project description:Although accumulating evidence indicates that both ?-catenin and osterix (Osx) are essential for bone and tooth development, few studies have investigated the interaction of these two key proteins in the context of cementogenesis. In this study, we used transgenic mice with constitutively active ?-catenin and inactive Osx in the dental mesenchyme to address this question. We found that cementoblasts with constitutively active ?-catenin require Osx to produce excessive cellular cementum, and that ablation of Osx prevents this abnormal accumulation. Importantly, cementoblasts transduced with retrovirus expressing constitutively active ?-catenin exhibited upregulation of Osx expression through direct binding to the promoter region of Osx. Osx regulates Lef1 expression and consequently could regulate T-cell factor/lymphoid enhancer factor (Tcf/Lef) binding activity in Wnt/?-catenin signaling. However, the loss of Tcf/Lef binding activity by Osx ablation was not rescued by transduction of retrovirus expressing constitutively active ?-catenin or ectopic Lef1 overexpression. These results suggest that the Tcf/Lef binding activity of Wnt/?-catenin signaling is Osx-dependent during cementogenesis. Moreover, Osx differentially regulates the expression of various Tcf family members, suggesting that Osx regulates cementogenesis by utilizing various Tcf/Lef-dependent mechanisms. This is the first report to show that downstream Osx signaling through Tcf/Lefs is critical for cementogenesis.
Project description:RMRP, the RNA component of mitochondrial RNA processing endoribonuclease, is a non-coding RNA (ncRNA) part of the RNase MRP complex functioning in mitochondrial and ribosomal RNA processing. Even though various mutations in the RMRP gene are linked to developmental defects and pathogenesis, its relevance to cancer etiology has not been well established. Here we examined the expression of RMRP and found a significant increase in colorectal and breast cancer patient tissues. So we tested whether the oncogenic signaling pathways, Wnt/?-catenin and Hippo/YAP pathways, are relevant to the enhanced expression of RMRP in cancer cells because of the predicted ?-catenin/TCF and YAP/TBX5 elements in the upstream regions of the RMRP gene. As expected, Wnt signal activation significantly induced the RMRP transcription thru ?-catenin and YAP transcription factors. More importantly, YAP protein was critical for RMRP transcription by association to the proximal site near the transcription start site of the RMRP gene, a Pol III promoter, along with ?-catenin and TBX5 proteins. We propose that the interplay of Wnt and Hippo signaling pathways could regulate target genes, coding or non-coding, by the ?-catenin/YAP/TBX5 transcription complex in cancer cells.
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: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:T-cell factor/lymphoid enhancer factor (TCF/LEF) proteins regulate transcription by recruiting ?-catenin and its associated co-regulators. Whether TCF/LEFs also recruit more factors through independent, direct interactions is not well understood. Here we discover Ring Finger Protein 14 (RNF14) as a new binding partner for all TCF/LEF transcription factors. We show that RNF14 positively regulates Wnt signalling in human cancer cells and in an in vivo zebrafish model by binding to target promoters with TCF and stabilizing ?-catenin recruitment. RNF14 depletion experiments demonstrate that it is crucial for colon cancer cell survival. Therefore, we have identified a key interacting factor of TCF/?-catenin complexes to regulate Wnt gene transcription.
Project description:The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of ?-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to ?-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of ?-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of ?-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to ?-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the ?-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of ?-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling.
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:Studies have demonstrated cross talk between beta-catenin and peroxisome proliferator-activated receptor gamma (PPARgamma) signaling pathways. Specifically, activation of PPARgamma induces the proteasomal degradation of beta-catenin in cells that express an adenomatous polyposis coli-containing destruction complex. In contrast, oncogenic beta-catenin is resistant to such degradation and inhibits the expression of PPARgamma target genes. In the present studies, we demonstrate a functional interaction between beta-catenin and PPARgamma that involves the T-cell factor (TCF)/lymphocyte enhancer factor (LEF) binding domain of beta-catenin and a catenin binding domain (CBD) within PPARgamma. Mutation of K312 and K435 in the TCF/LEF binding domain of an oncogenic beta-catenin (S37A) significantly reduces its ability to interact with and inhibit the activity of PPARgamma. Furthermore, these mutations render S37A beta-catenin susceptible to proteasomal degradation in response to activation of PPARgamma. Mutation of F372 within the CBD (helices 7 and 8) of PPARgamma disrupts its binding to beta-catenin and significantly reduces the ability of PPARgamma to induce the proteasomal degradation of beta-catenin. We suggest that in normal cells, PPARgamma can function to suppress tumorigenesis and/or Wnt signaling by targeting phosphorylated beta-catenin to the proteasome through a process involving its CBD. In contrast, oncogenic beta-catenin resists proteasomal degradation by inhibiting PPARgamma activity, which requires its TCF/LEF binding domain.