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Conséquences fonctionnelles des mutations du gène SF3B1 dans les syndromes myélodysplasiques avec sidéroblastes en couronne

Abstract : Myelodysplastic syndromes with ring sideroblasts (MDS-RS) are clonal hematopoietic stem cell (HSC) disorders characterized by anemia, bone marrow (BM) erythroid dysplasia and systemic iron accumulation. Mitochondrial iron accumulation and apoptosis of mature erythroblasts cause ineffective erythropoiesis. Splicing factor gene SF3B1 is mutated in ~90% of MDS-RS. SF3B1 mediates U2 snRNP recruitment to the branch point (BP) by interacting with the intronic RNA on both sides of the BP. SF3B1 also interacts with nucleosomes located at exonic positions. The nuclear dynamics of SF3B1 is under the control of Fanconi anemia FANCD2 and FANCI proteins, suggesting coordination between DNA repair pathway and cotranscriptional splicing during S phase. In response to DNA damage, the BRCA1/SF3B1 complex facilitates the splicing of DNA repair genes. Functional consequences of SF3B1 mutations in MDS-RS are still unknown. We first assessed the consequences of SF3B1 gene mutations on gene expression and splicing by sequencing the transcriptome of the BM mononuclear cells (MNC). SF3B1 hotspot mutations induce cryptic 3’ splice site selection through use of a different BP, as previous studies in MDS and other SF3B1-driven cancers have already shown. We identified an alternative transcript of erythroferrone (ERFE) in MNC of MDS patients with SF3B1 mutation. ERFE has recently been described as a major erythroid regulator of hepcidin, a key hormone that is involved in the control of iron homeostasis in the body. The expression of the variant ERFE transcript was restricted to SF3B1-mutated erythroblasts and allows the monitoring of clonal erythropoiesis. ERFE neo-isoform (ERFE+12) accounts for an overall increase of ERFE expression in SF3B1MUT MDS. ERFE+12 is translated into a functional variant protein in SF3B1MUT erythroblasts. Plasma concentrations of both canonical and aberrant ERFE proteins are significantly more elevated in SF3B1MUT MDS compared to SF3B1WT MDS and inversely correlated to hepcidin/ferritin ratio. Thus, we identified a SF3B1-dependent and erythroid cell intrinsic mechanism of hyperferritinemia in MDS-RS. We next analysed the DNA replication and DNA repair processes in human SF3B1MUT primary erythroblasts and in murine CRISPR-Cas9 Sf3b1K700E G1E-ER4 proerythroblastic cell line. We confirmed that SF3B1MUT erythroblasts have an increased proliferation rate, but they enter into terminal erythroid differentiation more rapidly than healthy controls or SF3B1WT MDS. We reported an increased DNA replication dynamics, since the rate of DNA fiber elongation is accelerated in Sf3b1K700E G1E-ER4 cells. It has recently been suggested that high speed of fork progression induces DNA replication stress. Consistently, we observed the spontaneous accumulation of single-stranded DNA covered by replication protein A in actively dividing SF3B1MUT proerythroblast and basophilic erythroblasts. No DNA-double strand break and no fork collapse were observed in SF3B1MUT cells. Thus, SF3B1 mutations induce a replication stress without stalling forks and contribute to the development of a clonal erythropoiesis. Mechanistically, we hypothesize that replication forks encounter less obstacles in SF3B1-mutated compared to SF3B1-wildtype erythroblasts. To address this point, we will investigate the presence of R-loops at the promoter regions of selected genes.
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Sabrina Bondu. Conséquences fonctionnelles des mutations du gène SF3B1 dans les syndromes myélodysplasiques avec sidéroblastes en couronne. Hématologie. Université Sorbonne Paris Cité, 2019. Français. ⟨NNT : 2019USPCB002⟩. ⟨tel-02495356⟩

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