Characterization of a Pygo2 splicing variant in zebrafish and potential Pygo2 roles in mediating melanoma in the zebrafish animal model

Pygopus is an essential transcription co-activator with the β-Catenin (β-Cat) complex for the canonical-Wnt (c-Wnt) signaling pathway in Drosophila, while in mammalian cells it could work either within or independently of the c-Wnt pathway. With a highly conserved domain (PHD) in its C-terminus, Pygo2 binds to BCL-9, which in turn binds β-Cat and activates c-Wnt–dependent transcriptional regulation. Emerging data indicate its role in multiple cancers, in which it acts through the c-Wnt-dependent signaling pathway. The role of c-Wnt has been more ambiguous in the case of melanoma, and the role of Pygo2 in this cancer type has not been tested yet. Melanoma is a serious type of skin cancer that develops in the melanocytes and dedifferentiates these cells into neural crest-like cells. c-Wnt signaling is determinant for neural crest cells proper behaviors and differentiation into melanocyte, and up or down regulation of c-Wnt signaling, and/or low/high levels of nuclear β-Cat have been shown in some melanoma studies. Previously in our laboratory we had identified in the zebrafish a new splicing variant of pygo2, which lacks the Plant-Homeo-Domain (PHD), and my goal was to finish the characterization of this splicing variant in the context of c-Wnt signaling and set up, with molecular genetic approaches, the fishlines that can be used to analyze the role of Pygo2 in the context of a melanoma model in the zebrafish. Using the TOP-FLASH assay, I showed that Pygo2ΔPHD has a dual role in cooperating with β-Cat in activating the reporter gene, and this depended on the time of cell transfection. Similarly, I discovered that Pygo2ΔPHD has the opposite effect when working with Pygo1 or Pygo2 full-length on the same process; again, this was dependent on the length of time the cells were transfected with the plasmids. This transcriptional behavior correlates with the cellular localization of the transfected protein, in fact I showed Pygo2ΔPHD entered in the nucleus only at 3 days from transfection. I have also created a mutant of the pygo2 full-length gene variant in the zebrafish by means of CRISPR/Cas9-based technology, which can still generate Pygo2ΔPHD but not Pygo2 full-length. These fish can survive through development, reach adulthood, and are fertile. Furthermore, in a p53-/- genetic background, I established a zebrafish transgenic line that expresses the mutated form of BRAF in the melanocytes. The resulting fishline has a high chance of developing nevi and melanomas.