Common Fragile Sites (CFSs) are chromosome regions prone to breaks/constrictions. CFSs are characterized by late replication timing mainly due to A/T level which form secondary structures, to the paucity of active replication origins and to the presence of large genes in which may occur the collision between replication and transcription processes. An incomplete DNA duplication results in genomic instability. Thus, to investigate the relationship between replication and tissue specificity of fragility we induced the expression of CFSs by an inhibitor of DNA polymerase (aphidicolin) in MRC-5 and IMR-90, two human lung fibroblast cell lines respectively of 14 and 16 weeks old, to compare the cytogenetic localization of CFSs between the same tissue but derived from two distinct individuals and different stage of differentiation. The most expressed CFSs in both cell lines are: 1p31.1 on chr. 1, 3q13.3 on chr. 3 and 7q11.2 chr. 7. These sites have typical features of CFSs, included many large and late replicated genes. FISH (fFluorescent In Situ Hybridization) technique was performed to delimit the boundaries of chromosomic fragile regions and to determine their replication profiles in combination with immunofluorescence against BrdU (thymidine analogue) on interphase nuclei in normal and stressful conditions. For additional control, the same procedure was carry out on lymphocyte metaphases and nuclei to verify if these sites were really not fragile in this tissue although display a non-canonical replication timing, indicating that structural peculiarities impair the replicative fork. The results show an incomplete replication of alleles at the end of S-phase under stress condition in both tissues, which raise fragility in fibroblasts, but not in lymphocytes, revealing a strict correlation between the expression of CFSs and tissue specificity. These outcomes suggest a prominent role of DNA replication in promoting a strong chromosomal instability.
The potential of the project lies in establishing the mechanisms at the base of genome instability at Common Fragile Sites. The topics covered the implicit aspects ranging from basic molecular mechanisms to evaluation of their involvement in cancer and/or diseases development and their evolutionary degree in the human genome respect to others species. The aim is to better clarify if they have a key role in the genome plasticity. The proposed methodology could clearly and directly outline the contribution of the replication process in the expression of Common Fragile Sites not only at a tissue-specific level, previously studied, but also at distinct differentiation stages of the same tissue. It would allow us to understand the correlation between the CFSs expression and differentiation stages during the embryogenesis, which has never been analyzed and therefore demonstrated. Besides, it could be interesting and fascinating start to understand why these particular regions even though subjected to structural aberrations in several tumors and diseases, are evolutionarily well conserved in mammals, especially from mouse to human. The expected results of the proposed research have the potential to be highly informative both from the point of view of basic and applied research.