Molecular and functional characterization of TIS7/PC, an immediate early gene involved in wnt-signaling and regulation of epithelial polarity

The central aim of my laboratory is to understand how the complex three-dimensional organization of polarized epithelial cells is lost during early stages of carcinogenesis. As our main model system, we use a mouse mammary gland epithelial cell line, expressing an estrogen-inducible c-JunER fusion protein, in which reversible loss of epithelial polarity can be studied. Our results show that this transient loss of polarity has to be regulated by a fine-tuned interplay of intracellular signalling systems, rearrangement of extracellular contacts and the cytoskeletal- and protein transport machineries involved, and that these processes might be much more tightly interconnected than previously appreciated. We have identified the immediate early gene product TIS7/PC4 as a JunER target gene by PCR-based differential display [DD]. The protein localizes at the inner leaflet of the basolateral plasma membrane in the vicinity of adherens junctions. Upon hormone-induced loss of polarity in c-JunER cells, the protein is upregulated, translocates to the cytoplasm and then accumulates in the nucleus. In addition, the TIS7/PC4 gene product interacts with components of the Wnt-signalling pathway and might have a co-repressor function. Therefore, we postulate that TIS 7/PC4 could be a co-repressor of mesenchymal genes, necessary for the correct expression of the differentiated phenotype of an epithelial cell. To test the hypothesis. the following specific aims are proposed: 1) To investigate the role of TIS 7/PC4 in the regulation of epithelial differentiation we will generate stable and inducible mammary epithelial cell lines with wild type and mutated TIS 7/PC4. These lines will be carefully characterized for the expression of an epithelial or mesenchymal phenotype and will also represent a valuable source for the biochemical analysis of TIS 7/PC4 interactions. In addition we will generate recombinant Adenovirus carrying both wild type and mutated TIS 7/PC4 for transient transfection studies in fully polarized epithelium. 2) The overall organization of the Wnt-pathway in mammalians and Xenopus is remarkably conserved. Early Xenopus embryos provide an useful experimental system for functional studies of molecules interfering with Wnt- signaling. We will inject TIS 7/PC4 messenger RNA into early Xenopus embryos and will analyse the embyonic phenotypes achieved. For instance, when TIS7 is injected together with b -catenin into ventral blastomers we will investigate if the b -catenin induced secondary axis formation can be blocked by TIS 7/PC4. 3) To identify TIS 7/PC4 binding proteins we will follow a two-pronged approach. With two hybrid screen we can check for interactions with known proteins of the Wnt-signalling pathway as well as screen against a library for unknown binding partners. We will complement this search by analyzing TIS 7/PC4 protein complexes after immunoprecipitation with high-resolution 2-D gel electrophoresis in combination with microsequencing or tandem mass spectrometry.