First name
Lara
Last name
Sicouri
Year of Study
Research Center
Thesis Title
Tumor suppressor mutagenesis driven by DNA deaminase
Thesis Abstract
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.
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.
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