SOX9 and TCF transcription factors associate to mediate Wnt/ß-catenin target gene activation in colorectal cancer.

Activation of the Wnt/ß-catenin pathway regulates gene expression by promoting the formation of a ß-catenin-T-cell factor (TCF) complex on target enhancers. In addition to TCFs, other transcription factors interact with the Wnt/ß-catenin pathway at different levels to produce tissue-specific patterns of Wnt target gene expression. The transcription factor SOX9 potently represses many Wnt target genes by downregulating ß-catenin protein levels. Here, we find using colony formation and cell growth assays that SOX9 surprisingly promotes the proliferation of Wnt-driven colorectal cancer (CRC) cells. In contrast to how it indirectly represses Wnt targets, SOX9 directly co-occupies and activates multiple Wnt-responsive enhancers in CRC cells. Our examination of the binding site grammar of these enhancers shows the presence of TCF and SOX9 binding sites that are necessary for transcriptional activation. In addition, we identify a physical interaction between the DNA-binding domains of TCFs and SOX9 and show that TCF-SOX9 interactions are important for target gene regulation and CRC cell growth. Our work demonstrates a highly context-dependent effect of SOX9 on Wnt targets, with the presence or absence of SOX9-binding sites on Wnt-regulated enhancers determining whether they are directly activated or indirectly repressed by SOX9.

Wnt target enhancer regulation by a CDX/TCF transcription factor collective and a novel DNA motif.

Transcriptional regulation by Wnt signalling is primarily thought to be accomplished by a complex of ß-catenin and TCF family transcription factors (TFs). Although numerous studies have suggested that additional TFs play roles in regulating Wnt target genes, their mechanisms of action have not been investigated in detail. We characterised a Wnt-responsive element (WRE) downstream of the Wnt target gene Axin2 and found that TCFs and Caudal type homeobox (CDX) proteins were required for its activation. Using a new separation-of-function TCF mutant, we found that WRE activity requires the formation of a TCF/CDX complex. Our systematic mutagenesis of this enhancer identified other sequences essential for activation by Wnt signalling, including several copies of a novel CAG DNA motif. Computational and experimental evidence indicates that the TCF/CDX/CAG mode of regulation is prevalent in multiple WREs. Put together, our results demonstrate the complex nature of cis- and trans- interactions required for signal-dependent enhancer activity.

Repression of Wnt/ß-catenin signaling by SOX9 and Mastermind-like transcriptional coactivator 2.

Wnt/ß-catenin signaling requires inhibition of a multiprotein destruction complex that targets ß-catenin for proteasomal degradation. SOX9 is a potent antagonist of the Wnt pathway and has been proposed to act through direct binding to ß-catenin or the ß-catenin destruction complex. Here, we demonstrate that SOX9 promotes turnover of ß-catenin in mammalian cell culture, but this occurs independently of the destruction complex and the proteasome. This activity requires SOX9's ability to activate transcription. Transcriptome analysis revealed that SOX9 induces the expression of the Notch coactivator Mastermind-like transcriptional activator 2 (MAML2), which is required for SOX9-dependent Wnt/ß-catenin antagonism. MAML2 promotes ß-catenin turnover independently of Notch signaling, and MAML2 appears to associate directly with ß-catenin in an in vitro binding assay. This work defines a previously unidentified pathway that promotes ß-catenin degradation, acting in parallel to established mechanisms. SOX9 uses this pathway to restrict Wnt/ß-catenin signaling.

The Wnt Transcriptional Switch: TLE Removal or Inactivation?

Many targets of the Wnt/ß-catenin signaling pathway are regulated by TCF transcription factors, which play important roles in animal development, stem cell biology, and oncogenesis. TCFs can regulate Wnt targets through a "transcriptional switch," repressing gene expression in unstimulated cells and promoting transcription upon Wnt signaling. However, it is not clear whether this switch mechanism is a general feature of Wnt gene regulation or limited to a subset of Wnt targets. Co-repressors of the TLE family are known to contribute to the repression of Wnt targets in the absence of signaling, but how they are inactivated or displaced by Wnt signaling is poorly understood. In this mini-review, we discuss several recent reports that address the prevalence and molecular mechanisms of the Wnt transcription switch, including the finding of Wnt-dependent ubiquitination/inactivation of TLEs. Together, these findings highlight the growing complexity of the regulation of gene expression by the Wnt pathway.

Wnt target genes and where to find them.

Wnt/ß-catenin signaling is highly conserved throughout metazoans, is required for numerous essential events in development, and serves as a stem cell niche signal in many contexts. Misregulation of the pathway is linked to several human pathologies, most notably cancer. Wnt stimulation results in stabilization and nuclear import of ß-catenin, which then acts as a transcriptional co-activator. Transcription factors of the T-cell family (TCF) are the best-characterized nuclear binding partners of ß-catenin and mediators of Wnt gene regulation. This review provides an update on what is known about the transcriptional activation of Wnt target genes, highlighting recent work that modifies the conventional model. Wnt/ß-catenin signaling regulates genes in a highly context-dependent manner, and the role of other signaling pathways and TCF co-factors in this process will be discussed. Understanding Wnt gene regulation has served to elucidate many biological roles of the pathway, and we will use examples from stem cell biology, metabolism, and evolution to illustrate some of the rich Wnt biology that has been uncovered.

The matrix protein Tiggrin regulates plasmatocyte maturation in Drosophila larva.

The lymph gland (LG) is a major source of hematopoiesis during Drosophila development. In this tissue, prohemocytes differentiate into multiple lineages, including macrophage-like plasmatocytes, which comprise the vast majority of mature hemocytes. Previous studies have uncovered genetic pathways that regulate prohemocyte maintenance and some cell fate choices between hemocyte lineages. However, less is known about how the plasmatocyte pool of the LG is established and matures. Here, we report that Tiggrin, a matrix protein expressed in the LG, is a specific regulator of plasmatocyte maturation. Tiggrin mutants exhibit precocious maturation of plasmatocytes, whereas Tiggrin overexpression blocks this process, resulting in a buildup of intermediate progenitors (IPs) expressing prohemocyte and hemocyte markers. These IPs likely represent a transitory state in prohemocyte to plasmatocyte differentiation. We also found that overexpression of Wee1 kinase, which slows G2/M progression, results in a phenotype similar to Tiggrin overexpression, whereas String/Cdc25 expression phenocopies Tiggrin mutants. Further analysis revealed that Wee1 inhibits plasmatocyte maturation through upregulation of Tiggrin transcription. Our results elucidate connections between the extracellular matrix and cell cycle regulators in the regulation of hematopoiesis.

The Role of the C-Clamp in Wnt-Related Colorectal Cancers.

T-cell Factor/Lymphoid Enhancer Factor (TCF/LEF) transcription factors are major regulators of Wnt targets, and the products of the TCF7 and TCF7L2 genes have both been implicated in the progression of colorectal cancer in animal models and humans. TCFs recognize specific DNA sequences through their high mobility group (HMG) domains, but invertebrate TCFs and some isoforms of vertebrate TCF7 and TCF7L2 contain a second DNA binding domain known as the C-clamp. This review will cover the basic properties of C-clamps and their importance in Wnt signaling, using data from Drosophila, C. elegans, and mammalian cell culture. The connection between C-clamp containing TCFs and colorectal cancer will also be discussed.

Bipartite recognition of DNA by TCF/Pangolin is remarkably flexible and contributes to transcriptional responsiveness and tissue specificity of wingless signaling.

The T-cell factor (TCF) family of transcription factors are major mediators of Wnt/ß-catenin signaling in metazoans. All TCFs contain a High Mobility Group (HMG) domain that possesses specific DNA binding activity. In addition, many TCFs contain a second DNA binding domain, the C-clamp, which binds to DNA motifs referred to as Helper sites. While HMG and Helper sites are both important for the activation of several Wnt dependent cis-regulatory modules (W-CRMs), the rules of what constitutes a functional HMG-Helper site pair are unknown. In this report, we employed a combination of in vitro binding, reporter gene analysis and bioinformatics to address this question, using the Drosophila family member TCF/Pangolin (TCF/Pan) as a model. We found that while there were constraints for the orientation and spacing of HMG-Helper pairs, the presence of a Helper site near a HMG site in any orientation increased binding and transcriptional response, with some orientations displaying tissue-specific patterns. We found that altering an HMG-Helper site pair from a sub-optimal to optimal orientation/spacing dramatically increased the responsiveness of a W-CRM in several fly tissues. In addition, we used the knowledge gained to bioinformatically identify two novel W-CRMs, one that was activated by Wnt/ß-catenin signaling in the prothoracic gland, a tissue not previously connected to this pathway. In sum, this work extends the importance of Helper sites in fly W-CRMs and suggests that the type of HMG-Helper pair is a major factor in setting the threshold for Wnt activation and tissue-responsiveness.

Wnt-mediated repression via bipartite DNA recognition by TCF in the Drosophila hematopoietic system.

The Wnt/ß-catenin signaling pathway plays many important roles in animal development, tissue homeostasis and human disease. Transcription factors of the TCF family mediate many Wnt transcriptional responses, promoting signal-dependent activation or repression of target gene expression. The mechanism of this specificity is poorly understood. Previously, we demonstrated that for activated targets in Drosophila, TCF/Pangolin (the fly TCF) recognizes regulatory DNA through two DNA binding domains, with the High Mobility Group (HMG) domain binding HMG sites and the adjacent C-clamp domain binding Helper sites. Here, we report that TCF/Pangolin utilizes a similar bipartite mechanism to recognize and regulate several Wnt-repressed targets, but through HMG and Helper sites whose sequences are distinct from those found in activated targets. The type of HMG and Helper sites is sufficient to direct activation or repression of Wnt regulated cis-regulatory modules, and protease digestion studies suggest that TCF/Pangolin adopts distinct conformations when bound to either HMG-Helper site pair. This repressive mechanism occurs in the fly lymph gland, the larval hematopoietic organ, where Wnt/ß-catenin signaling controls prohemocytic differentiation. Our study provides a paradigm for direct repression of target gene expression by Wnt/ß-catenin signaling and allosteric regulation of a transcription factor by DNA.

Distinct DNA binding sites contribute to the TCF transcriptional switch in C. elegans and Drosophila.

Regulation of gene expression by signaling pathways often occurs through a transcriptional switch, where the transcription factor responsible for signal-dependent gene activation represses the same targets in the absence of signaling. T-cell factors (TCFs) are transcription factors in the Wnt/ß-catenin pathway, which control numerous cell fate specification events in metazoans. The TCF transcriptional switch is mediated by many co-regulators that contribute to repression or activation of Wnt target genes. It is typically assumed that DNA recognition by TCFs is important for target gene location, but plays no role in the actual switch. TCF/Pangolin (the fly TCF) and some vertebrate TCF isoforms bind DNA through two distinct domains, a High Mobility Group (HMG) domain and a C-clamp, which recognize DNA motifs known as HMG and Helper sites, respectively. Here, we demonstrate that POP-1 (the C. elegans TCF) also activates target genes through HMG and Helper site interactions. Helper sites enhanced the ability of a synthetic enhancer to detect Wnt/ß-catenin signaling in several tissues and revealed an unsuspected role for POP-1 in regulating the C. elegans defecation cycle. Searching for HMG-Helper site clusters allowed the identification of a new POP-1 target gene active in the head muscles and gut. While Helper sites and the C-clamp are essential for activation of worm and fly Wnt targets, they are dispensable for TCF-dependent repression of targets in the absence of Wnt signaling. These data suggest that a fundamental change in TCF-DNA binding contributes to the transcriptional switch that occurs upon Wnt stimulation.

Structure-function analysis of the C-clamp of TCF/Pangolin in Wnt/ß-catenin signaling.

The evolutionarily conserved Wnt/ß-catenin (Wnt/ß-cat) pathway plays an important role in animal development in metazoans. Many Wnt targets are regulated by members of the TCF/LEF1 (TCF) family of transcription factors. All TCFs contain a High Mobility Group (HMG) domain that bind specific DNA sequences. Invertebrate TCFs and some vertebrate TCF isoforms also contain another domain, called the C-clamp, which allows TCFs to recognize an additional DNA motif known as the Helper site. While the C-clamp has been shown to be important for regulating several Wnt reporter genes in cell culture, its physiological role in regulating Wnt targets is less clear. In addition, little is known about this domain, except that two of the four conserved cysteines are functionally important. Here, we carried out a systematic mutagenesis and functional analysis of the C-clamp from the Drosophila TCF/Pangolin (TCF/Pan) protein. We found that the C-clamp is a zinc-binding domain that is sufficient for binding to the Helper site. In addition to this DNA-binding activity, the C-clamp also inhibits the HMG domain from binding its cognate DNA site. Point mutations were identified that specifically affected DNA-binding or reduced the inhibitory effect. These mutants were characterized in TCF/Pan rescue assays. The specific DNA-binding activity of the C-clamp was essential for TCF/Pan function in cell culture and in patterning the embryonic epidermis of Drosophila, demonstrating the importance of this C-clamp activity in regulating Wnt target gene expression. In contrast, the inhibitory mutation had a subtle effect in cell culture and no effect on TCF/Pan activity in embryos. These results provide important information about the functional domains of the C-clamp, and highlight its importance for Wnt/ß-cat signaling in Drosophila.

Celebrating 30 years of Wnt signaling.

Wnt signal transduction is crucial for embryonic development and tissue homeostasis in multicellular animals. Hyperactivation of the Wnt pathway drives tumor formation, yet activation of the Wnt pathway in stem cells holds great promise for injury repair and regeneration. Between 27 June and 1 July 2012, scientists from all over the globe gathered in the beachfront town of Egmond aan Zee in the Netherlands to celebrate the 30th anniversary of this blossoming and exciting field. The latest advances and breakthroughs were discussed at the aptly named European Molecular Biology Organization conference 30 Years of Wnt Signalling. Many presenters discussed unpublished data, a hallmark of past and hopefully future Wnt meetings. This Meeting Report summarizes some of the highlights of this conference, including the presentation of the long-awaited crystal structure of a Wnt protein bound to its receptor and the identification of exciting new possibilities for targeting the pathway in treating disease.

TCF/LEFs and Wnt signaling in the nucleus.

T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors are the major end point mediators of Wnt/Wingless signaling throughout metazoans. TCF/LEFs are multifunctional proteins that use their sequence-specific DNA-binding and context-dependent interactions to specify which genes will be regulated by Wnts. Much of the work to define their actions has focused on their ability to repress target gene expression when Wnt signals are absent and to recruit ß-catenin to target genes for activation when Wnts are present. Recent advances have highlighted how these on/off actions are regulated by Wnt signals and stabilized ß-catenin. In contrast to invertebrates, which typically contain one TCF/LEF protein that can both activate and repress Wnt targets, gene duplication and isoform complexity of the family in vertebrates have led to specialization, in which individual TCF/LEF isoforms have distinct activities.

TCFs and Wnt/ß-catenin signaling: more than one way to throw the switch.

Wnts are conserved, secreted signaling proteins that can influence cell behavior by stabilizing ß-catenin. Accumulated ß-catenin enters the nucleus, where it physically associates with T-cell factor (TCF) family members to regulate target gene expression in many developmental and adult tissues. Recruitment of ß-catenin to Wnt response element (WRE) chromatin converts TCFs from transcriptional repressors to activators. This review will outline the complex interplay between factors contributing to TCF repression and coactivators working with ß-catenin to regulate Wnt targets. In addition, three variations of the standard transcriptional switch model will be discussed. One is the Wnt/ß-catenin symmetry pathway in Caenorhabditis elegans, where Wnt-mediated nuclear efflux of TCF is crucial for activation of targets. Another occurs in vertebrates, where distinct TCF family members are associated with repression and activation, and recent evidence suggests that Wnt signaling facilitates a "TCF exchange" on WRE chromatin. Finally, a "reverse switch" mechanism for target genes that are directly repressed by Wnt/ß-catenin signaling occurs in Drosophila cells. The diversity of TCF regulatory mechanisms may help to explain how a small group of transcription factors can function in so many different contexts to regulate target gene expression.

The microRNA miR-8 is a positive regulator of pigmentation and eclosion in Drosophila.

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally silence gene expression by binding to target mRNAs. Previous studies have identified the miRNA miR-8 as a pleiotropic regulator of Drosophila development, controlling body size and neuronal survival by targeting multiple mRNAs. In this study we demonstrate that miR-8 is also required for proper spatial patterning of pigment on the adult abdominal cuticle in females but not males. RESULTS: Female adult flies lacking miR-8 exhibit decreased pigmentation of the dorsal abdomen, with a pattern of pigmentation similar to wild type flies grown at higher temperatures. This pigmentation defect in miR-8 mutants is independent of the previously reported body size defect, and miR-8 acts directly in the developing cuticle to regulate pigmentation patterning. The decrease in pigmentation in miR-8 mutants was more pronounced in flies grown at higher temperatures. We also found that loss of miR-8 dramatically affected the ability to eclose at higher temperatures. CONCLUSION: Loss of miR-8 increased the sensitivity of Drosophila to higher temperatures for both pigmentation patterning and the ability to eclose. Together, these data suggest that miR-8 acts as a buffer to stabilize gene expression patterns in the midst of environmental variation.

The oligomeric state of CtBP determines its role as a transcriptional co-activator and co-repressor of Wingless targets.

C-terminal-binding protein (CtBP) is a well-characterized transcriptional co-repressor that requires homo-dimerization for its activity. CtBP can both repress and activate Wingless nuclear targets in Drosophila. Here, we examine the role of CtBP dimerization in these opposing processes. CtBP mutants that cannot dimerize are able to promote Wingless signalling, but are defective in repressing Wingless targets. To further test the role of dimerization in repression, the positions of basic and acidic residues that form inter-molecular salt bridges in the CtBP dimerization interface were swapped. These mutants cannot homo-dimerize and are compromised for repression. However, their co-expression leads to hetero-dimerization and consequent repression of Wingless targets. Our results support a model where CtBP is a gene-specific regulator of Wingless signalling, with some targets requiring CtBP dimers for inhibition while other targets utilize CtBP monomers for activation of their expression. Functional interactions between CtBP and Pygopus, a nuclear protein required for Wingless signalling, support a model where monomeric CtBP acts downstream of Pygopus in activating some Wingless targets.

Drosophila ptip is essential for anterior/posterior patterning in development and interacts with the PcG and trxG pathways.

Development of the fruit fly Drosophila depends in part on epigenetic regulation carried out by the concerted actions of the Polycomb and Trithorax group of proteins, many of which are associated with histone methyltransferase activity. Mouse PTIP is part of a histone H3K4 methyltransferase complex and contains six BRCT domains and a glutamine-rich region. In this article, we describe an essential role for the Drosophila ortholog of the mammalian Ptip (Paxip1) gene in early development and imaginal disc patterning. Both maternal and zygotic ptip are required for segmentation and axis patterning during larval development. Loss of ptip results in a decrease in global levels of H3K4 methylation and an increase in the levels of H3K27 methylation. In cell culture, Drosophila ptip is required to activate homeotic gene expression in response to the derepression of Polycomb group genes. Activation of developmental genes is coincident with PTIP protein binding to promoter sequences and increased H3K4 trimethylation. These data suggest a highly conserved function for ptip in epigenetic control of development and differentiation.

Wnt signaling from development to disease: insights from model systems.

One of the early surprises in the study of cell adhesion was the discovery that beta-catenin plays dual roles, serving as an essential component of cadherin-based cell-cell adherens junctions and also serving as the key regulated effector of the Wnt signaling pathway. Here, we review our current model of Wnt signaling and discuss how recent work using model organisms has advanced our understanding of the roles Wnt signaling plays in both normal development and in disease. These data help flesh out the mechanisms of signaling from the membrane to the nucleus, revealing new protein players and providing novel information about known components of the pathway.