TUMOR SUPPRESSOR MUTAGENESIS DRIVEN BY DNA DEAMINASE

Genomic instability is commonly associated with pathological disorders including cancer. The progressive accumulation of genetic abnormalities in cancer-associated genes can confer cellular autonomous proliferation, contributing to the oncogenic transformation. Although animal models have been instrumental to the understanding of the molecular mechanisms responsible for tumor progression, there are severe limitations for success. This thesis work aimed at the development of an innovative animal model that could recapitulate the ongoing lifelong accumulation of DNA lesions, leading to neoplastic transformation. To this end, the natural DNA mutating enzyme AID was fused to sequence specific DNA binding proteins. They were engineered to target tumor suppressor genes and to induce low-frequency mutagenesis in cell lines and in zebrafish. A TALE-Aid fusion protein was targeted to the p53 locus of mouse cell lines, and the Aid-dependent mutations were monitored by next-generation sequencing. The induced mutations occurred at a comparable frequency to those observed in Aid-induced non-immunoglobulin gene targets in B cells. Mutations were found mostly in the DNA binding domain of p53, possibly reflecting AID- hotspot residues in p53. Our approach also induced mutations that have not been characterized previously, and could provide further insight into p53 dependent oncogenesis. When TALE-AID were expressed and targeted to the p53 locus of zebrafish embryos, the activity induced developmental abnormalities with variable severity, leading to both an increased mortality, and impaired ovarian maturation and fertility. We have developed and initially characterized a novel tool for the in vitro / in vivo study of the accumulation of mutations in cancer-associated genes.