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ERC

FAtoUnFRAGILITY Report Summary

Project ID: 638898
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - FAtoUnFRAGILITY (Fanconi anemia : a disease model to understand causes and consequences of common fragile site instability.)

Reporting period: 2015-06-01 to 2016-11-30

Summary of the context and overall objectives of the project

Replication of DNA 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 characteristic. It is therefore essential to identify the factors preventing its appearance but also the molecular bases 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 therefore raised great interest from the scientific community and today we know more about the factors leading to the instability of these regions. Replication, gene transcription, and chromatin conformation in these regions have been identified as the main factors regulating CFS stability. Despite these discoveries, several points remain to be clarified, notably 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 instability of CFS. Indeed, CFSs are particularly sensitive in this disease and many aberrations observed in patients’ cells are localized at the level of CFS. We now know that proteins defective in FA (which form the FANC pathway) 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 from the beginning of the project to the end of the period covered by the report and main results achieved so far

Task 1)
In the first 18 months from the beginning 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, and of FANC deficient mouse cells compared with the wt counterparts, as part of the aim 1 of the project. 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 region. We have then analyzed the histone modifications that characterize these regions, in the presence or absence of replication stress, and their dependency on a functional FANC pathway. These experiments allowed us to set up the conditions in order to carry out ChIP experiments followed by high-throughput sequencing. Analysis of ChIP-seq data is currently ongoing. We have also set up the conditions to perform the analysis of the replication program in FANC-depleted compared to control cells, in collaboration with Dr JC Cadoret (Jacques Monod Institute, Paris). Most of the work has been performed by the PhD student Philippe Fernandes. In addition, Michail Fragkos, a post-doc hired on the project, has contributed to the writing of an invited review currently in press (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)

In the first 18 months of the project, we have set up the mouse colony and started the comparative genomic and transcriptomic analysis of hematopoietic cells from wild type (WT) and Fanca knockout (KO) mice, in order to identify early genetic and epigenetic alterations associated to the loss of a functional FANC pathway. This work has been performed by a research engineer, Emilie Dassé, hired on the project after the renouncement of P Pawlikowska, with support from the Genomic and Bioinformatic Platforms of Gustave Roussy. Analysis and validation of the obtained results is ongoing. Based on the obtained preliminary results, we are also pursuing the characterization of molecular factors involved in replication stress response and in the process of megakaryopoieisis, which is dysfunctional in FA, in collaboration with Dr W Vainchenker (Gustave Roussy). As part of the aim 2 of the project, we have asked the authorization and we are currently planning the establishment of double mutant mouse models to study the functional interaction of FANC pathway with candidate CFS genes.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

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. Based on this premise, we expect to identify actionable molecular targets to be used in preventive or therapeutic strategies.
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