Phd in GENETICS AND MOLECULAR BIOLOGY

Thesis title: Common Fragile Sites: a new tool to study chromosome instability diseases

Replication stress is a major cause of Chromosomal Instability (CIN) that manifests as chromosome rearrangements, gaps and breaks, including those cytologically expressed within specific chromosome regions named Common Fragile Sites (CFSs). The molecular mechanisms of CFSs instability have not been completely elucidated yet. In the first part of my work, I characterized the expression and the replication timing of human CFSs upon treatment with aphidicolin (APH ), a DNA polymerase α (alpha) inhibitor , in three cellular lines: Glioblastoma Multiforme U-251 MG cell line and two isogenic Fanconi Anemia lymphoblastoid lines (the mutated HSC72 FA-A and the corrected HSC72 FANCA). GBM and FA cell lines are both associated with high physiological levels of CIN and thus are good genetic models to understand the causes underlying CFSs instability. Glioblastoma Multiforme (GBM) is a tumor of the Central Nervous System (CNS) and Fanconi Anemia (FA) is a rare multigenic disorder caused by mutations in FA DNA repair genes. I identified CFSs that showed a frequency equal to at least 1% of the total gaps/breaks: 17 CFSs in GBM, 16 CFSs in HSC72 FA-A, 19 CFSs in HSC72 FANCA. Only few of them were found to be cell type-specific. In the last part of my work, CFSs induced by 4', 6′-diamidino-2-phenylindole hydrochloride (DAPI), a DNA dye binding to AT-rich sequences and acting as an under-condensing agent in G2-phase, were analyzed in a pathological background such as FA cells (which are characterized by a prolonged G2-phase upon DNA damage) to understand how the post-replicative chromatin compaction is essential to their integrity. Presence of long genes, incomplete replication, improper chromatin condensation and DNA synthesis during mitosis (MiDAS) after APH and DAPI treatment suggest that impaired replication process and defective chromatin compaction may contribute to the loci-specific fragility in U-251 MG cells and in both HSC72 FA lymphoblasts cell lines. Altogether, my work offers a comprehensive characterization of CFSs expressed in GBM and FA cells that may be further exploited for cytogenetic and clinical studies to advance our understanding of the physiological status and these genic and genetic disorders.