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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - FAtoUnFRAGILITY (Fanconi anemia : a disease model to understand causes and consequences of common fragile site instability.)

Teaser

Summary

DNA replication is an essential process that must be strictly regulated during cell proliferation. It is during this key stage that genomic instability can arise and participate in the development of cancer, of which it is a hallmark. Therefore, it is essential to identify the factors preventing its appearance but also the molecular mechanisms leading to this instability. Some regions of the genome are particularly sensitive to problems occurring during replication, namely, common fragile sites (CFSs). These regions are frequently altered in the early stages of cancer, which causes the loss of tumor suppressor genes or the amplification of oncogenes, thus participating in tumor development. The basis of this instability has raised great interest from the scientific community and today we know more about the factors underlying the instability of these regions. Replication, transcription, and chromatin conformation of the genomic regions encompassing CFSs have been identified as the main factors regulating their stability. Despite these discoveries, several points remain to be clarified, notably the role of the proteins involved in maintaining the stability of these regions and their importance in the pathological process. Our project employs Fanconi Anemia (FA), a rare genetic disease characterized by a high predisposition of patients to cancer, as a model for studying the causes and the consequences of the instability of CFSs. Indeed, many aberrations observed in patientsâ€™ cells are localized at the level of CFSs. We know now that the proteins constituting the FANC pathway (deficient in FA) are necessary to maintain the stability of CFSs, but the mechanisms by which these proteins perform this function remain to be elucidated. The main objectives of this project are:

1) To elucidate how the FANC proteins regulate CFS loci, and
2) To analyze the mechanistic link between CFS damage and the FA pathological phenotypes.

Understanding these mechanisms would be beneficial not only to design preventive or therapeutic strategies for FA patients but also to combat cancer in the general population.

Work performed

\"Task 1)

In the first part of the project, we have set up the laboratory and the conditions to carry out gene expression, chromatin and cytogenetic analysis of human cells depleted of FANC proteins compared with control cells. In addition, we have validated the FANCD2 antibody for chromatin immunoprecipitation (ChIP) and then carried out ChIP experiments in human cells followed by qPCR analysis with different pairs of primers covering the most fragile CFS regions. These experiments allowed us to set up the conditions in order to carry out ChIP experiments followed by high-throughput sequencing. We have then analyzed the genomic localization of FANCD2, a key protein in the FANC pathway, and how its localization is affected following replicative stress. We have also analyzed the histone modifications that characterize these regions, in the presence or absence of replication stress, and their dependency on a functional FANC pathway. In addition, we have investigated how the expression of genes at CFS that are targeted by FANCD2 is regulated. We have then started to characterize signaling pathways that may be involved in CFS regulation and how they are modulated by the FANC pathway, as well as novel factors that can impact their fragility and affect cell proliferation and homeostasis.
Most of the work has been performed by a PhD student Philippe Fernandes. A manuscript including these results is currently in preparation. In addition, Michail Fragkos, a post-doc hired on the project, has contributed to the writing of an invited review recently published in Cell Cycle (Fragkos and Naim, Cell Cycle 2017) and extended the project framework to the analysis of the interaction of FANC pathway with another molecular pathway involved in maintenance of CFS stability. This work, in collaboration with Dr A Coquelle (Institute of Cancer Research of Montpellier), is expected to be published in the next few months.

Task 2)

The first part of the project has been devoted to the establishment of the mouse colony and setting up the experiments necessary to perform the comparative genomic and transcriptomic analysis of hematopoietic cells from wild type (WT) and Fanca knockout (KO) mice, in order to identify early events and genomic alterations associated to the loss of a functional FANC pathway. This work has been performed by a research engineer, Emilie DassÃ©, hired on the project, and successively by another research engineer, Yidan Wang, with support from the Genomics and Bioinformatics Platforms of Gustave Roussy. Analysis and validation of the results is ongoing. Based on the obtained preliminary results, we are pursuing the identification and characterization of molecular factors and signaling networks deregulated in Fanca KO mice, and proceeding to the functional validation of the identified deregulated genes/pathways. As part of the aim 2 of the project, we have asked the authorization and planned to establish double mutant mouse models in order to study the functional interaction of FANC pathway with candidate genes participating in the identified pathways. In addition, the PI has established a collaboration in order to test the expression of selected genes in FA patients, which could help in designing new diagnostic or therapeutic strategies.

The preliminary results of the research project have been presented by the PI and/or team members in several national and international meetings, such as the 10th quinquennial Conference on Response of DNA damage in Holland, the Fanconi Anemia Research Fund (FARF) Symposium in Seattle (USA), the DNA replication meeting \"\"At the Intersection of DNA Replication and Genome Maintenance: from Mechanisms to Therapy\"\" in Trieste (Italy), or the Replication-Recombination-Repair (3R) Meeting in Giens (France). In addition, the PI has been invited by collaborators and other colleagues to give seminars and present the project in academic institutions.
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Final results

In recent years, several studies have highlighted the prominent role of DNA replication stress in driving genomic instability, a hallmark of cancer, neurodegenerative diseases and aging. Elucidating how cells respond to replication stress and what are the events involved in replication-stress-driven genetic and epigenetic instability is fundamental for our understanding of disease mechanisms. FA is a chromosomal instability and cancer predisposition syndrome, and the molecular pathway deficient in this disease is involved in replication stress response and genome maintenance. Therefore, FA is an ideal disease model to uncover the molecular bases underlying genome instability and cancer predisposition. We have started to identify and characterize the mechanisms and (epi)genetic factors underlying CFS instability, as well as a potential function of CFSs in regulating cellular homeostasis. Understanding the cellular response to replication stress could help to design strategies to prevent genomic instability in normal and precancerous cells on one side and, on the other, to exacerbate replication stress in tumor cells to kill them. Based on this premise, we expect to identify actionable molecular targets that can be used for preventive or therapeutic interventions.