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 β-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

The chromatin remodelers ISWI and ACF1 directly repress Wingless transcriptional targets

AbstractThe 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

Receptor endocytosis: Frizzled joins the ubiquitin club

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102106/1/embj2010132.pd

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 β-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

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

The C-clamp is required for patterning of the <i>Drosophila</i> embryonic epidermis.

<p>(A) Crossing scheme used to generate embryos containing a P[<i>Da</i>-Gal4] driver and P[UAS-<i>TCF/Pan</i>] transgene in a <i>TCF²/TCF³</i> transheterozygous mutant background. UAS-<i>TCF/Pan</i> (UAS-<i>TCF</i>) encodes for either wild type or a mutant <i>TCF/Pan</i>. (B–C) Darkfield micrographs of the ventral cuticle control (B) or P[<i>Da</i>-Gal4]/+; <i>TCF²/TCF³</i> embryos showing the normal and <i>TCF/Pan</i> mutant phenotypes, respectively. (D–G) Cuticles of <i>TCF²/TCF³</i> mutants expressing wild-type (D), mutant 5 (E), mutant 4 (F) or mutant 8 (G) <i>TCF/Pan</i> cDNAs. (H) Western blots showing comparable levels of TCF/Pan (upper blot) expression for WT and various C-clamp mutants in P[<i>Da</i>-Gal4]; P[UAS-<i>TCF/Pan</i>] embryos with Tubulin used as the loading control (lower blot).</p

Recombinant HMG-C-clamp fragment contains near stoichiometric amounts of Zinc.

<p>Recombinant HMG-C-clamp and HMG domain proteins were purified from <i>E. coli</i> and subjected to ICP-MS. See Materials and Methods for details of ICP-MS analysis.</p