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Chabalier-Taste C.,Toulouse 1 University Capitole | Brichese L.,Toulouse 1 University Capitole | Racca C.,CNRS Institute of Pharmacology and Structural Biology | Racca C.,Toulouse 1 University Capitole | And 7 more authors.
Oncotarget | Year: 2016

Accurate repair of DNA double-strand breaks (DSB) caused during DNA replication and by exogenous stresses is critical for the maintenance of genomic integrity. There is growing evidence that the Polo-like kinase 1 (Plk1) that plays a number of pivotal roles in cell proliferation can directly participate in regulation of DSB repair. In this study, we show that Plk1 regulates BRCA1, a key mediator protein required to efficiently repair DSB through homologous recombination (HR). Following induction of DSB, BRCA1 concentrates in distinctive large nuclear foci at damage sites where multiple DNA repair factors accumulate. First, we found that inhibition of Plk1 shortly before DNA damage sensitizes cells to ionizing radiation and reduces DSB repair by HR. Second, we provide evidence that BRCA1 foci formation induced by DSB is reduced when Plk1 is inhibited or depleted. Third, we identified BRCA1 as a novel Plk1 substrate and determined that Ser1164 is the major phosphorylation site for Plk1 in vitro. In cells, mutation of Plk1 sites on BRCA1 significantly delays BRCA1 foci formation following DSB, recapitulating the phenotype observed upon Plk1 inhibition. Our data then assign a key function to Plk1 in BRCA1 foci formation at DSB, emphasizing Plk1 importance in the HR repair of human cells. Source


Di Paolo A.,CNRS Institute of Pharmacology and Structural Biology | Di Paolo A.,University Paul Sabatier | Racca C.,CNRS Institute of Pharmacology and Structural Biology | Racca C.,University Paul Sabatier | And 5 more authors.
FASEB Journal | Year: 2014

In contrast to its well-known role in the DNA damage response during interphase, the function of BRCA1 in the maintenance of chromosomal stability during mitosis remains to be defined. In this study, we uncover a novel role of BRCA1 in preserving centromere integrity in mitotic human cells. Using immunofluorescence and chromatin immunoprecipitation approaches, we report BRCA1 association with centromeric chromatin during mitosis. BRCA1 depletion impairs centromeric cohesion, leading to an increase in interkinetochore distance and in unpaired sister-chromatids frequency during prometaphase. Moreover, BRCA1 loss partially decreased accumulation of the Aurora B kinase at the centromere. We found that proper recruitment of the DNMT3b DNA methyltransferase to satellite sequences is BRCA1-dependent during mitosis, suggesting that DNA hypomethylation contributes to Aurora B mislocalization. BRCA1-deficient cells exhibited decreased ability to correct improper Aurora B-dependent chromosome-spindle attachments and to align chromosomes at metaphase. Finally, we show that BRCA1 disruption promotes merotelic kinetochore attachments that represent a major mechanism of aneuploidy in human cells. In summary, we report here a novel function of BRCA1 in maintaining chromosomal stability through its contribution to the mitotic centromere integrity necessary for faithful segregation of sister-chromatids during cell division. © FASEB. Source


Roos-Weil D.,French Institute of Health and Medical Research | Roos-Weil D.,University Paris Saclay | Roos-Weil D.,Equipe labellisee Ligue Nationale Contre le Cancer | Nguyen-Khac F.,French Institute of Health and Medical Research | And 4 more authors.
American Journal of Hematology | Year: 2016

Recent advances in massively parallel sequencing technologies have provided a detailed picture of the mutational landscape in CLL and underscored the vast degree of interpatient and intratumor heterogeneities. These studies have led to the characterization of novel putative driver genes and recurrently affected biological pathways, and to the modeling of CLL clonal evolution. We herein review selected aspects including recent advances in the biology of CLL and present cellular and biological processes involved in the development of CLL and potentially other mature B-cell lymphoproliferative neoplasms. © 2016 Wiley Periodicals, Inc. Source


Sahin U.,Institute Pasteur Paris | Sahin U.,French Institute of Health and Medical Research | Sahin U.,Equipe labellisee Ligue Nationale Contre le Cancer | Sahin U.,University Paris Diderot | And 10 more authors.
PLoS ONE | Year: 2014

Gene silencing by small RNAs has emerged as a powerful post-transcriptional regulator of gene expression, however processes underlying regulation of the small RNA pathway in vivo are still largely elusive. Here, we identified sumoylation as a novel post-translational modification acting on Ago2, the main effector of small RNA-mediated gene silencing. We demonstrate that Ago2 can be modified by SUMO1 and SUMO2/3 and identified Lys402 as the major Ago2 sumoylation site in vivo. Ago2 physically interacts with the SUMO E2 conjugating enzyme Ubc9 and the E3 ligase RanBP2 facilitates Ago2 sumoylation in vitro. Mutation of Lys402 enhances the stability of Ago2 protein and impairment of cellular sumoylation by siRNA- or shRNA-mediated extinction of Ubc9 or in Ubc9 knockout mouse tissues results in increased steady-state levels and enhanced stability of Ago2. Similarly, knockdown of RanBP2 or of the SAE2 E1 enzyme enhances Ago2 protein levels. Lys402 is located in the L2g1 loop linking the PAZ and PIWI domains of Ago2, in the immediate vicinity of Tyr393 which can be phosphorylated, implying that the L2g1 linker represents an easily accessible hot spot for post-translational modifications. Altogether, our results show that sumoylation of Ago2 at Lys402 negatively regulates its stability, thereby establishing a first link between SUMO and the small RNA machinery. © 2014 Sahin et al. Source


Fritah S.,Institute Pasteur Paris | Fritah S.,French Institute of Health and Medical Research | Fritah S.,Equipe labellisee Ligue Nationale Contre le Cancer | Fritah S.,CRP Sante | And 15 more authors.
EMBO Reports | Year: 2014

Shigella flexneri, the etiological agent of bacillary dysentery, invades the human colonic epithelium and causes its massive inflammatory destruction. Little is known about the post-translational modifications implicated in regulating the host defense pathway against Shigella. Here, we show that SUMO-2 impairs Shigella invasion of epithelial cells in vitro. Using mice haploinsufficient for the SUMO E2 enzyme, we found that sumoylation regulates intestinal permeability and is required to restrict epithelial invasion and control mucosal inflammation. Quantitative proteomics reveals that Shigella infection alters the sumoylation status of a restricted set of transcriptional regulators involved in intestinal functions and inflammation. Consistent with this, sumoylation restricts the pro-inflammatory transcriptional response of Shigella-infected guts. Altogether, our results show that the SUMO pathway is an essential component of host innate protection, as it reduces the efficiency of two key steps of shigellosis: invasion and inflammatory destruction of the intestinal epithelium. © 2014 The Authors. Source

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