"Task 1)
Thanks to the ERC grant, we have set up the laboratory and all the conditions necessary to carry out gene expression, chromatin and cytogenetic analyses of cells depleted of FANC proteins compared with control cells. We have performed chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing to analyze the genomic localization of FANCD2, a key protein in the FANC pathway, and investigated how the expression of CFS genes is regulated. We have identified in the FANCD2 DNA binding sites, so-called MURE sequences (mitochondrial unfolded protein response element). We have found that CFS genes respond to the mitochondrial unfolded protein response (mtUPR), a signaling pathway triggered by the presence of unfolded or misfolded proteins in the mitochondria. Activation of the mtUPR promotes the recruitment of FANCD2 and its partner FANCI that attenuate their transcription and avoid or resolve transcription-replication conflicts, preserving genome stability and mitochondrial homeostasis.
Most of this work was performed by the PhD student Philippe Fernandes and published as a preprint (Fernandes P et al., bioRxiv 2019 808915). In addition, with Michail Fragkos, a post-doc hired on the project, we wrote a review article in which we describe the mechanisms allowing rescue from replication stress during mitosis (Fragkos and Naim, Cell Cycle 2017). Together with Viviana Barra (another post-doc in the team), Michalis found an interaction between the Dicer pathway (involved in microRNA biogenesis) and FANCD2 in maintenance of CFS stability. This work, in collaboration with Dr A. Coquelle (Institute of Cancer Research of Montpellier), was published in Oncotarget (Fragkos M et al., Oncotarget, 2019). More recently, in a review written with Maha Said (PhD student in the team) and Therese Wilhelm (former post-doc in the team), we discuss how replication stress leads to both structural and numerical chromosomal instability (Wilhelm T et al., Genes, 2020). The PI also contributed to a work in collaboration with the team of PH Gaillard (Cancer Research Center of Marseille), showing that interaction between SLX4 (FANCP) and the helicase RTEL1 drives the assembly of FANCD2 foci in the vicinity of RNA polymerase II, to prevent transcription-mediated replication stress (Takedachi A et al., Nat Struct Mol Biol, 2020).
Task 2)
The second aim of the project was devoted to establish the mouse colony and set 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 the research engineers hired on the project, Emilie Dassé and successively Yidan Wang, with support from the Genomics and Bioinformatics Platforms of Gustave Roussy. Interestingly, we have identified genes that are differentially expressed in Fanca KO hematopoietic cells specifically in elderly mice. Based on these results, we have set up collaborations with Dr Enrico Cappelli (Gaslini Hospital, Genoa) and Pr Silvia Ravera (University of Genoa) in order to test the expression of these genes in FA patients, which could help to develop new diagnostic and/or prognostic biomarkers of disease progression, and orient therapeutic strategies. Thanks to the results of the ERC project and to collaboration with Dr Cappelli and Pr Ravera, we have provided evidence that the passage from the hypoxic bone marrow niche to the bloodstream triggers the metabolic dysfunction of mononuclear cells isolated from FA patients (Cappelli E et al., Redox Biology, 2020), confirming the impact of mitochondrial metabolism on FA cell homeostasis.
The results of the ERC project have been presented by the PI and team members at national and international conferences and meetings, such as the Fanconi Anemia Symposium in Chicago in 2019 (V. Naim), the Cell Symposium ""Multifaceted Mitochondria"" in San Diego in 2018 (P. Fernandes), the 3R meeting (Replication-Repair-Recombination) in Giens in 2017 (P. Fernandes), the meeting ""At the intersection of DNA replication and genome maintenance: from mechanisms to therapy"" in Trieste in 2016 (M. Fragkos), or the International Symposium ""DNA repair targeting as cancer therapy"" in Villejuif in 2016 (V. Naim). In addition, the PI has been invited by collaborators and other colleagues to give seminars and to present the project in several research and academic institutions."
