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.

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 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.

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.

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.

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.

Novel TCF-binding sites specify transcriptional repression by Wnt signalling.

Both transcriptional activation and repression have essential functions in maintaining proper spatial and temporal control of gene expression. Although Wnt signalling is often associated with gene activation, we have identified several directly repressed targets of Wnt signalling in Drosophila. Here, we explore how individual Wnt target genes are specified for signal-induced activation or repression. Similar to activation, repression required binding of Armadillo (Arm) to the N terminus of TCF. However, TCF/Arm mediated repression by binding to DNA motifs that are markedly different from typical TCF-binding sites. Conversion of the novel motifs to standard TCF-binding sites reversed the mode of regulation, resulting in Wnt-mediated activation instead of repression. A mutant form of Arm defective in activation was still functional for repression, indicating that distinct domains of the protein are required for each activity. This study suggests that the sequence of TCF-binding sites allosterically regulates the TCF/Arm complex to effect either transcriptional activation or repression.

The microRNA miR-8 is a conserved negative regulator of Wnt signaling.

Wnt signaling plays many important roles in animal development. This evolutionarily conserved signaling pathway is highly regulated at all levels. To identify regulators of the Wnt/Wingless (Wg) pathway, we performed a genetic screen in Drosophila. We identified the microRNA miR-8 as an inhibitor of Wg signaling. Expression of miR-8 potently antagonizes Wg signaling in vivo, in part by directly targeting wntless, a gene required for Wg secretion. In addition, miR-8 inhibits the pathway downstream of the Wg signal by repressing TCF protein levels. Another positive regulator of the pathway, CG32767, is also targeted by miR-8. Our data suggest that miR-8 potently antagonizes the Wg pathway at multiple levels, from secretion of the ligand to transcription of target genes. In addition, mammalian homologues of miR-8 promote adipogenesis of marrow stromal cells by inhibiting Wnt signaling. These findings indicate that miR-8 family members play an evolutionarily conserved role in regulating the Wnt signaling pathway.

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.

Spenito and Split ends act redundantly to promote Wingless signaling.

Wingless (Wg)/Wnt signaling directs a variety of cellular processes during animal development by promoting the association of Armadillo/beta-catenin with TCFs on Wg-regulated enhancers (WREs). Split ends (Spen), a nuclear protein containing RNA recognition motifs (RRMs) and a SPOC domain, is required for optimal Wg signaling in several fly tissues. In this report, we demonstrate that Spenito (Nito), the only other fly protein containing RRMs and a SPOC domain, acts together with Spen to positively regulate Wg signaling. The partial defect in Wg signaling observed with spen RNAi was enhanced by simultaneous knockdown of nito while it was rescued by expression of nito in wing imaginal discs. In cell culture, depletion of both factors causes a greater defect in the activation of several Wg targets than RNAi of either spen or nito alone. These nuclear proteins are not required for Armadillo stabilization or the recruitment of TCF and Armadillo to a WRE. Loss of Wg target gene activation in cells depleted for spen and nito was not dependent on the transcriptional repressor Yan or Suppressor of Hairless, two previously identified targets of Spen. We propose that Spen and Nito act redundantly downstream of TCF/Armadillo to activate many Wg transcriptional targets.

Regulation of the feedback antagonist naked cuticle by Wingless signaling.

Signaling pathways usually activate transcriptional targets in a cell type-specific manner. Notable exceptions are pathway-specific feedback antagonists, which serve to restrict the range or duration of the signal. These factors are often activated by their respective pathways in a broad array of cell types. For example, the Wnt ligand Wingless (Wg) activates the naked cuticle (nkd) gene in all tissues examined throughout Drosophila development. How does the nkd gene respond in such an unrestricted manner to Wg signaling? Analysis in cell culture revealed regions of the nkd locus that contain Wg response elements (WREs) that are directly activated by the pathway via the transcription factor TCF. In flies, Wg signaling activates these WREs in multiple tissues, in distinct but overlapping patterns. These WREs are necessary and largely sufficient for nkd expression in late stage larval tissues, but only contribute to part of the embryonic expression pattern of nkd. These results demonstrate that nkd responsiveness to Wg signaling is achieved by several WREs which are broadly (but not universally) activated by the pathway. The existence of several WREs in the nkd locus may have been necessary to allow the Wg signaling-Nkd feedback circuit to remain intact as Wg expression diversified during animal evolution.

The chromatin remodelers ISWI and ACF1 directly repress Wingless transcriptional targets.

The highly conserved Wingless/Wnt signaling pathway controls many developmental processes by regulating the expression of target genes, most often through members of the TCF family of DNA-binding proteins. In the absence of signaling, many of these targets are silenced, by mechanisms involving TCFs that are not fully understood. Here we report that the chromatin remodeling proteins ISWI and ACF1 are required for basal repression of WG target genes in Drosophila. This regulation is not due to global repression by ISWI and ACF1 and is distinct from their previously reported role in chromatin assembly. While ISWI is localized to the same regions of Wingless target gene chromatin as TCF, we find that ACF1 binds much more broadly to target loci. This broad distribution of ACF1 is dependent on ISWI. ISWI and ACF1 are required for TCF binding to chromatin, while a TCF-independent role of ISWI-ACF1 in repression of Wingless targets is also observed. Finally, we show that Wingless signaling reduces ACF1 binding to WG targets, and ISWI and ACF1 regulate repression by antagonizing histone H4 acetylation. Our results argue that WG signaling activates target gene expression partly by overcoming the chromatin barrier maintained by ISWI and ACF1.

Drosophila split ends homologue SHARP functions as a positive regulator of Wnt/beta-catenin/T-cell factor signaling in neoplastic transformation.

Wnt ligands have pleiotropic and context-specific roles in embryogenesis and adult tissues. Among other effects, certain Wnts stabilize the beta-catenin protein, leading to the ability of beta-catenin to activate T-cell factor (TCF)-mediated transcription. Mutations resulting in constitutive beta-catenin stabilization underlie development of several human cancers. Genetic studies in Drosophila highlighted the split ends (spen) gene as a positive regulator of Wnt-dependent signaling. We have assessed the role of SHARP, a human homologue of spen, in Wnt/beta-catenin/TCF function in mammalian cells. We found that SHARP gene and protein expression is elevated in human colon and ovarian endometrioid adenocarcinomas and mouse colon adenomas and carcinomas carrying gene defects leading to beta-catenin dysregulation. When ectopically expressed, the silencing mediator for retinoid and thyroid receptors/histone deacetylase 1-associated repressor protein (SHARP) protein potently enhanced beta-catenin/TCF transcription of a model reporter gene and cellular target genes. Inhibition of endogenous SHARP function via RNA inhibitory (RNAi) approaches antagonized beta-catenin/TCF-mediated activation of target genes. The effect of SHARP on beta-catenin/TCF-regulated genes was mediated via a functional interaction between SHARP and TCF. beta-Catenin-dependent neoplastic transformation of RK3E cells was enhanced by ectopic expression of SHARP, and RNAi-mediated inhibition of endogenous SHARP in colon cancer cells inhibited their transformed growth. In toto, our findings implicate SHARP as an important positive regulator of Wnt signaling in cancers with beta-catenin dysregulation.

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.