Kenneth M. Cadigan, Ph.D. - US grants

Signaling between neighboring cells is crucial for many cell fate decisions during development. Surprisingly, the same secreted signals are often used in different tissues to elicit apparently unrelated cellular responses. Our goal is to understand better the molecular mechanisms underlying this phenomenon, using a relatively simple example of tissue-specific regulation by the Drosophila secreted protein Wingless (Wg). In the developing fly wing, Wg is required for the formation of sensory bristles. However, in the eye Wg signaling prevents cells from adopting a bristle cell fate. The bristle precursor cells in both tissues receive the Wg signal, but they interpret the signal differently. To learn more about the tissue-specific aspects of Wg signaling, two approachers are proposed. We have identified the daughterless (da) gene as potential direct target of Wg-dependent bristle inhibition in the eye. We will determine the molecular mechanism of this regulation and whether Da is a target of Wg signaling in the wing. The second approach is to characterize two genes whose mutant phenotypes indicate that they play important roles in tissue-specific Wg signaling. One gene encodes a protein kinase that is required for Wg regulation of bristle formation in the eye but not in the wing. Genetic epitasis analysis as well as biochemical characterization of the kinase will determine its relationship to known components of the Wg signaling pathway. The second gene acts as a positive effector of Wg signaling in the eye, but it antagonizes the pathway in the wing. This gene will be cloned and sequenced to elucidate the role it plays in Wg signaling. We expect that the two approaches will converge on a model to explain the tissue- specific control of bristle formation by Wg. Wg is one of the best characterized members of a large family of secreted proteins (known as Wnts) conserved throughout the animal kingdom. Wnts have been found to play many important roles in the development of both invertebrates and vertebrates. In addition, Wnt signaling has been implicated in tumorigenesis in mice and humans. Since the machinery for Wnt signal transduction is also evolutionarily conserved, understanding tissue specific mechanisms of Wg signaling in flies should have broad relevance to other organisms.

DESCRIPTION (provided by applicant): Cells receive and interpret signals from their extracellular environment through biochemical cascades known as signal transduction pathways. These pathways control many aspects of cell growth and differentiation. Mutations activating these processes are often found in human cancers. An important example of this is found in the Wnt signaling pathway. Wnts are secreted glycoproteins commonly used during invertebrate and vertebrate development that function through a highly conserved signaling apparatus. Recessive mutations in negative regulators of the pathway and gain-of-function mutations in positive effecters play a causal role in several cancers. Our goal is to identify new Wnt signaling components, by studying Wingless (Wg), a well characterized Drosophila Wnt. In one approach, a genetic screen was performed which identified a gene, gammy legs (gam), which blocks Wg signaling when over expressed. Removal of gam activity also leads to loss of Wg signaling. gam encodes a protein with a putative nuclear localization sequence and a PHD finger. The molecular mechanism of Gam action in Wg signaling will be persued using genetic epistasis in flies and fly cell culture, identification of possible binding partners for the Gam protein and a systematic structure/function mutational analysis. Two predicted human genes that have signficant sequence similarity to gam have also been identified in the database. These genes will be cloned and analyzed to determine if they play an important role in Wnt signaling in human cell lines. The second approach takes advantage of the recent finding that incubating Drosophila cells with dsRNA corresponding to a particular gene specify targets that gene's mRNA for degradation. This technology will be used to screen potential fly homologs of several factors recently implicated in vertebrate Wnt signaling through protein-protein interaction screens. If reduction of a gene's expression causes Wg signaling to be effected in cell culture, then fly mutants lacking these genes will be isolated and analyzed for Wg signaling defects. Obtaining genetic confirmation of the importance of these putative Wnt signaling components will provide the confidence to pursue their study in the context of cancer biology and as targets for drug discovery.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Secreted glycoproteins of the Wnt family act through an evolutionarily conserved signaling cascade which promotes the nuclear accumulation of beta-catenin, which interacts with members of the TCF family of DNA-binding proteins to activate target gene transcription. TCFs binds to specific sequences, but the consensus is so loose that potential TCF binding sites can be found throughout the genome. Despite this, we find that TCF is bound to specific locations in Wnt targets, corresponding to Wnt response elements (WREs). Systematic mutagenesis of a WRE revealed the presence of additional motifs that act with TCF binding sites to mediate activation by Wnt signaling. These motifs (called Helper sites) are found in several other WREs, and genome-wide searches for conserved TCF/Helper site clusters have identified other putative WREs. The functional significance of the Helper sites in these elements will be tested. The molecular mechanism of how Helper sites interact with TCF binding sites will be explored, and the trans-acting factor(s) that bind to it will be characterized. Binding of beta-catenin to TCF converts it from a transcriptional repressor to an activator. We have found that Wnt signaling promotes histone acetylation throughout target loci, which is correlated with activation of transcription. This widespread modification appears to be required to antagonize the action of factors that silence Wnt targets. The mechanisms and relationship between these processes will be explored in detail. Our data indicates that the transcriptional switch at Wnt targets involves changes in chromatin structure far beyond what was previously recognized. PROJECT NARRATIVE: The Wnt signaling pathway plays important roles in cell fate decisions during development, and is required for the maintenance of stem cell populations in adult tissues. Misregulation of the pathway plays a causal role in many human cancers. Our studies to understand the role of motifs besides TCF sites that contribute to WRE function will lead to better bioinformatic methods to identify Wnt targets in many important biological contexts. Our work on the role of chromatin modifications in regulating the TCF transcriptional switch will serve as a paradigm to study these processes in mammalian systems. [unreadable] [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Wnts are secreted proteins that regulate cell behavior through several pathways, the best characterized of which is Wnt/ß-catenin signaling, which plays many important roles in animal development and adult tissue homeostasis. For example, in intestinal crypts, the pathway is required to maintain stem cell populations, and is also needed to specify Paneth cells, which secrete anti-microbial proteins to keep gut bacteria in check. Misregulation of the pathway is associated with colorectal cancer (CRC) and inflammatory bowel disorders. Wnt signaling promotes nuclear accumulation of ß-catenin, which is then recruited to Wnt responsive cis- regulatory modules (W-CRMs) by members of the TCF/LEF1 (TCF) family of transcription factors (TFs). Once there, ß-catenin acts as a potent activator of Wnt target gene transcription. Several genome-wide studies have linked TCF occupancy with other TFs in the genome. This clustering of TFs has been termed a TF collective. However, the mechanisms underlying functional interactions between TCFs and other TFs are poorly understood. We have characterized two W-CRMs from the human axin2 and c-myc genes in detail using cell culture. The c-myc W-CRM is of interest because it contains a polymorphism in a TCF site linked to increased colorectal cancer (CRC) in humans. Our preliminary data support a model where TCFs work with several other TFs to achieve Wnt activation of these W-CRMs. We will characterize the physical and functional interactions between these factors, to understand how a TF collective containing TCFs operates. We have found that the c-myc W-CRM is active in several tissues in mouse embryos and given its link to CRC in humans, we will examine its activity in the adult mouse intestine. We will also test whether the factors important for activating this W-CRM in cell culture are also required in mouse tissues. One of these factors is Sox9, a TF which is which is typically thought of as an antagonist of Wnt/ß-catenin signaling, but which also cooperates with TCFs to activate the c-myc W-CRM. This cooperation may explain why TCFs and Sox9 are both required for Paneth cell formation in the intestine. In addition, Sox9 is required for testis formation and XY individuals with Sox9 mutations often develop as females. We are characterizating Sox9 mutants that specifically affect its ability to act with or against Wnt signaling. We propose to engineer mice with these mutations, to determine whether these activities underlie its role in Paneth cell specification and sex determination. This work will increase our understanding of the role of Wnt signaling in CRC as well as inflammatory bowel disorders that affect Paneth cell function.