Instabilité génétique et Cancérogenèse

Publications de l’équipe

Année de publication : 2020

(2020 Aug 26)

Identification and Analysis of Different Types of UFBs

Methods Mol Biol. : DOI : 10.1007/978-1-0716-0644-5_13 En savoir plus

Ultrafine anaphase bridges (UFBs) result from a defect in sister chromatid segregation during anaphase. They arise from particular DNA structures, mostly generated at specific loci in the human genome, such as centromeres, common fragile sites, telomeres, or ribosomal DNA. Increases in UFB frequency are a marker of genetic instability, and their detection has become a classic way of detecting such genetic instability over the last decade. Here we describe a protocol to stain different types of UFBs in adherent human cells.

Sandra Cunha Silveira, Géraldine Buhagiar‑Labarchède, Rosine Onclercq‑Delic, Simon Gemble, Elias Bou Samra, Hamza Mameri, Patricia Duchambon, Christelle Machon, Jérôme Guitton & Mounira Amor‑Guéret (2020 Aug 17)

A decrease in NAMPT activity impairs basal PARP-1 activity in cytidine deaminase deficient-cells, independently of NAD+

Scientific Reports : 10 : 13907 : DOI : 10.1038/s41598-020-70874-6 En savoir plus

Cytidine deaminase (CDA) deficiency causes pyrimidine pool disequilibrium. We previously reported that the excess cellular dC and dCTP resulting from CDA deficiency jeopardizes genome stability, decreasing basal poly(ADP-ribose) polymerase 1 (PARP-1) activity and increasing ultrafine anaphase bridge (UFB) formation. Here, we investigated the mechanism underlying the decrease in PARP-1 activity in CDA-deficient cells. PARP-1 activity is dependent on intracellular NAD+ concentration. We therefore hypothesized that defects of the NAD+ salvage pathway might result in decreases in PARP-1 activity. We found that the inhibition or depletion of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD+ salvage biosynthesis pathway, mimicked CDA deficiency, resulting in a decrease in basal PARP-1 activity, regardless of NAD+ levels. Furthermore, the expression of exogenous wild-type NAMPT fully restored basal PARP-1 activity and prevented the increase in UFB frequency in CDA-deficient cells. No such effect was observed with the catalytic mutant. Our findings demonstrate that (1) the inhibition of NAMPT activity in CDA-proficient cells lowers basal PARP-1 activity, and (2) the expression of exogenous wild-type NAMPT, but not of the catalytic mutant, fully restores basal PARP-1 activity in CDA-deficient cells; these results strongly suggest that basal PARP-1 activity in CDA-deficient cells decreases due to a reduction of NAMPT activity.

Simon Gemble, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Gaëlle Fontaine, Sarah Lambert, Mounira Amor-Guéret (2020 May 14)

Topoisomerase IIα prevents ultrafine anaphase bridges by two mechanisms.

Open biology : 190259 : DOI : 10.1098/rsob.190259 En savoir plus

Topoisomerase IIα (Topo IIα), a well-conserved double-stranded DNA (dsDNA)-specific decatenase, processes dsDNA catenanes resulting from DNA replication during mitosis. Topo IIα defects lead to an accumulation of ultrafine anaphase bridges (UFBs), a type of chromosome non-disjunction. Topo IIα has been reported to resolve DNA anaphase threads, possibly accounting for the increase in UFB frequency upon Topo IIα inhibition. We hypothesized that the excess UFBs might also result, at least in part, from an impairment of the prevention of UFB formation by Topo IIα. We found that Topo IIα inhibition promotes UFB formation without affecting the global disappearance of UFBs during mitosis, but leads to an aberrant UFB resolution generating DNA damage within the next G1. Moreover, we demonstrated that Topo IIα inhibition promotes the formation of two types of UFBs depending on cell cycle phase. Topo IIα inhibition during S-phase compromises complete DNA replication, leading to the formation of UFB-containing unreplicated DNA, whereas Topo IIα inhibition during mitosis impedes DNA decatenation at metaphase-anaphase transition, leading to the formation of UFB-containing DNA catenanes. Thus, Topo IIα activity is essential to prevent UFB formation in a cell-cycle-dependent manner and to promote DNA damage-free resolution of UFBs.


Année de publication : 2019

Matthieu Gratia, Mathieu P Rodero, Cécile Conrad, Elias Bou Samra, Mathieu Maurin, Gillian I Rice, Darragh Duffy, Patrick Revy, Florence Petit, Russell C Dale, Yanick J Crow, Mounira Amor-Gueret, Nicolas Manel (2019 Apr 1)

Bloom syndrome protein restrains innate immune sensing of micronuclei by cGAS.

The Journal of experimental medicine : DOI : jem.20181329 En savoir plus

Cellular innate immune sensors of DNA are essential for host defense against invading pathogens. However, the presence of self-DNA inside cells poses a risk of triggering unchecked immune responses. The mechanisms limiting induction of inflammation by self-DNA are poorly understood. BLM RecQ-like helicase is essential for genome integrity and is deficient in Bloom syndrome (BS), a rare genetic disease characterized by genome instability, accumulation of micronuclei, susceptibility to cancer, and immunodeficiency. Here, we show that BLM-deficient fibroblasts show constitutive up-regulation of inflammatory interferon-stimulated gene (ISG) expression, which is mediated by the cGAS-STING-IRF3 cytosolic DNA-sensing pathway. Increased DNA damage or down-regulation of the cytoplasmic exonuclease TREX1 enhances ISG expression in BLM-deficient fibroblasts. cGAS-containing cytoplasmic micronuclei are increased in BS cells. Finally, BS patients demonstrate elevated ISG expression in peripheral blood. These results reveal that BLM limits ISG induction, thus connecting DNA damage to cellular innate immune response, which may contribute to human pathogenesis.


Année de publication : 2017

Alexis Fouquin, Josée Guirouilh-Barbat, Bernard Lopez, Janet Hall, Mounira Amor-Guéret, Vincent Pennaneach (2017 Dec 1)

PARP2 controls double-strand break repair pathway choice by limiting 53BP1 accumulation at DNA damage sites and promoting end-resection.

Nucleic acids research : DOI : 10.1093/nar/gkx881 En savoir plus

Double strand breaks (DSBs) are one of the most toxic lesions to cells. DSB repair by the canonical non-homologous end-joining (C-EJ) pathway involves minor, if any, processing of the broken DNA-ends, whereas the initiation of DNA resection channels the broken-ends toward DNA repair pathways using various lengths of homology. Mechanisms that control the resection initiation are thus central to the regulation to the choice of DSB repair pathway. Therefore, understanding the mechanisms which regulate the initiation of DNA end-resection is of prime importance. Our findings reveal that poly(ADP-ribose) polymerase 2 (PARP2) is involved in DSBR pathway choice independently of its PAR synthesis activity. We show that PARP2 favors repair by homologous recombination (HR), single strand annealing (SSA) and alternative-end joining (A-EJ) rather than the C-EJ pathway and increases the deletion sizes at A-EJ junctions. We demonstrate that PARP2 specifically limits the accumulation of the resection barrier factor 53BP1 at DNA damage sites, allowing efficient CtIP-dependent DNA end-resection. Collectively, we have identified a new PARP2 function, independent of its PAR synthesis activity, which directs DSBs toward resection-dependent repair pathways.

Elias Bou Samra, Géraldine Buhagiar-Labarchède, Christelle Machon, Jérôme Guitton, Rosine Onclercq-Delic, Michael R Green, Olivier Alibert, Claude Gazin, Xavier Veaute, Mounira Amor-Guéret (2017 Sep 25)

A role for Tau protein in maintaining ribosomal DNA stability and cytidine deaminase-deficient cell survival.

Nature communications : 693 : DOI : 10.1038/s41467-017-00633-1 En savoir plus

Cells from Bloom’s syndrome patients display genome instability due to a defective BLM and the downregulation of cytidine deaminase. Here, we use a genome-wide RNAi-synthetic lethal screen and transcriptomic profiling to identify genes enabling BLM-deficient and/or cytidine deaminase-deficient cells to tolerate constitutive DNA damage and replication stress. We found a synthetic lethal interaction between cytidine deaminase and microtubule-associated protein Tau deficiencies. Tau is overexpressed in cytidine deaminase-deficient cells, and its depletion worsens genome instability, compromising cell survival. Tau is recruited, along with upstream-binding factor, to ribosomal DNA loci. Tau downregulation decreases upstream binding factor recruitment, ribosomal RNA synthesis, ribonucleotide levels, and affects ribosomal DNA stability, leading to the formation of a new subclass of human ribosomal ultrafine anaphase bridges. We describe here Tau functions in maintaining survival of cytidine deaminase-deficient cells, and ribosomal DNA transcription and stability. Moreover, our findings for cancer tissues presenting concomitant cytidine deaminase underexpression and Tau upregulation open up new possibilities for anti-cancer treatment.Cytidine deaminase (CDA) deficiency leads to genome instability. Here the authors find a synthetic lethal interaction between CDA and the microtubule-associated protein Tau deficiencies, and report that Tau depletion affects rRNA synthesis, ribonucleotide pool balance, and rDNA stability.

Simon Gemble, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Christian Jaulin, Mounira Amor-Guéret (2017 Jun 3)

Cytidine deaminase deficiency impairs sister chromatid disjunction by decreasing PARP-1 activity.

Cell cycle (Georgetown, Tex.) : 1-8 : DOI : 10.1080/15384101.2017.1317413 En savoir plus

Bloom Syndrome (BS) is a rare genetic disease characterized by high levels of chromosomal instability and an increase in cancer risk. Cytidine deaminase (CDA) expression is downregulated in BS cells, leading to an excess of cellular dC and dCTP that reduces basal PARP-1 activity, compromising optimal Chk1 activation and reducing the efficiency of downstream checkpoints. This process leads to the accumulation of unreplicated DNA during mitosis and, ultimately, ultrafine anaphase bridge (UFB) formation. BS cells also display incomplete sister chromatid disjunction when depleted of cohesin. Using a combination of fluorescence in situ hybridization and chromosome spreads, we investigated the possible role of CDA deficiency in the incomplete sister chromatid disjunction in cohesin-depleted BS cells. The decrease in basal PARP-1 activity in CDA-deficient cells compromised sister chromatid disjunction in cohesin-depleted cells, regardless of BLM expression status. The observed incomplete sister chromatid disjunction may be due to the accumulation of unreplicated DNA during mitosis in CDA-deficient cells, as reflected in the changes in centromeric DNA structure associated with the decrease in basal PARP-1 activity. Our findings reveal a new function of PARP-1 in sister chromatid disjunction during mitosis.

Hamza Mameri, Ivan Bieche, Dider Meseure, Elisabetta Marangoni, Géraldine Buhagiar-Labarchède, Andre Nicolas, Sophie Vacher, Rosine Onclercq-Delic, Vinodh Rajapakse, Sudhir Varma, William C Reinhold, Yves Pommier, Mounira Amor-Guéret (2017 Apr 15)

Cytidine deaminase deficiency reveals new therapeutic opportunities against cancer.

Clinical cancer research : an official journal of the American Association for Cancer Research : DOI : 10.1158/1078-0432.CCR-16-0626 En savoir plus

One of the main challenges in cancer therapy is the identification of molecular mechanisms mediating resistance or sensitivity to treatment. Cytidine deaminase (CDA) was reported to be downregulated in cells derived from patients with Bloom syndrome, a genetic disease associated with a strong predisposition to a wide range of cancers. The purpose of this study was to determine whether CDA deficiency could be associated with tumors from the general population and could constitute a predictive marker of susceptibility to anti-tumor drugs.


Année de publication : 2016

Simon Gemble, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Denis Biard, Sarah Lambert, Mounira Amor-Guéret (2016 Aug 15)

A balanced pyrimidine pool is required for optimal Chk1 activation to prevent ultrafine anaphase bridge formation.

Journal of cell science : 3167-77 : DOI : 10.1242/jcs.187781 En savoir plus

Cytidine deaminase (CDA) deficiency induces an excess of cellular dCTP, which reduces basal PARP-1 activity, thereby compromising complete DNA replication, leading to ultrafine anaphase bridge (UFB) formation. CDA dysfunction has pathological implications, notably in cancer and in Bloom syndrome. It remains unknown how reduced levels of PARP-1 activity and pyrimidine pool imbalance lead to the accumulation of unreplicated DNA during mitosis. We report that a decrease in PARP-1 activity in CDA-deficient cells impairs DNA-damage-induced Chk1 activation, and, thus, the downstream checkpoints. Chemical inhibition of the ATR-Chk1 pathway leads to UFB accumulation, and we found that this pathway was compromised in CDA-deficient cells. Our data demonstrate that ATR-Chk1 acts downstream from PARP-1, preventing the accumulation of unreplicated DNA in mitosis, and, thus, UFB formation. Finally, delaying entry into mitosis is sufficient to prevent UFB formation in both CDA-deficient and CDA-proficient cells, suggesting that both physiological and pathological UFBs are derived from unreplicated DNA. Our findings demonstrate an unsuspected requirement for a balanced nucleotide pool for optimal Chk1 activation both in unchallenged cells and in response to genotoxic stress.


Année de publication : 2015

Sara Chiker, Vincent Pennaneach, Damarys Loew, Florent Dingli, Denis Biard, Fabrice P Cordelières, Simon Gemble, Sophie Vacher, Ivan Bieche, Janet Hall, Marie Fernet (2015 Aug 3)

Cdk5 promotes DNA replication stress checkpoint activation through RPA-32 phosphorylation, and impacts on metastasis free survival in breast cancer patients.

Cell cycle (Georgetown, Tex.) : 3066-78 : DOI : 10.1080/15384101.2015.1078020 En savoir plus

Cyclin dependent kinase 5 (Cdk5) is a determinant of PARP inhibitor and ionizing radiation (IR) sensitivity. Here we show that Cdk5-depleted (Cdk5-shRNA) HeLa cells show higher sensitivity to S-phase irradiation, chronic hydroxyurea exposure, and 5-fluorouracil and 6-thioguanine treatment, with hydroxyurea and IR sensitivity also seen in Cdk5-depleted U2OS cells. As Cdk5 is not directly implicated in DNA strand break repair we investigated in detail its proposed role in the intra-S checkpoint activation. While Cdk5-shRNA HeLa cells showed altered basal S-phase dynamics with slower replication velocity and fewer active origins per DNA megabase, checkpoint activation was impaired after a hydroxyurea block. Cdk5 depletion was associated with reduced priming phosphorylations of RPA32 serines 29 and 33 and SMC1-Serine 966 phosphorylation, lower levels of RPA serine 4 and 8 phosphorylation and DNA damage measured using the alkaline Comet assay, gamma-H2AX signal intensity, RPA and Rad51 foci, and sister chromatid exchanges resulting in impaired intra-S checkpoint activation and subsequently higher numbers of chromatin bridges. In vitro kinase assays coupled with mass spectrometry demonstrated that Cdk5 can carry out the RPA32 priming phosphorylations on serines 23, 29, and 33 necessary for this checkpoint activation. In addition we found an association between lower Cdk5 levels and longer metastasis free survival in breast cancer patients and survival in Cdk5-depleted breast tumor cells after treatment with IR and a PARP inhibitor. Taken together, these results show that Cdk5 is necessary for basal replication and replication stress checkpoint activation and highlight clinical opportunities to enhance tumor cell killing.

Simon Gemble, Akshay Ahuja, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Julien Dairou, Denis S F Biard, Sarah Lambert, Massimo Lopes, Mounira Amor-Guéret (2015 Jul 16)

Pyrimidine Pool Disequilibrium Induced by a Cytidine Deaminase Deficiency Inhibits PARP-1 Activity, Leading to the Under Replication of DNA.

PLoS genetics : e1005384 : DOI : 10.1371/journal.pgen.1005384 En savoir plus

Genome stability is jeopardized by imbalances of the dNTP pool; such imbalances affect the rate of fork progression. For example, cytidine deaminase (CDA) deficiency leads to an excess of dCTP, slowing the replication fork. We describe here a novel mechanism by which pyrimidine pool disequilibrium compromises the completion of replication and chromosome segregation: the intracellular accumulation of dCTP inhibits PARP-1 activity. CDA deficiency results in incomplete DNA replication when cells enter mitosis, leading to the formation of ultrafine anaphase bridges between sister-chromatids at « difficult-to-replicate » sites such as centromeres and fragile sites. Using molecular combing, electron microscopy and a sensitive assay involving cell imaging to quantify steady-state PAR levels, we found that DNA replication was unsuccessful due to the partial inhibition of basal PARP-1 activity, rather than slower fork speed. The stimulation of PARP-1 activity in CDA-deficient cells restores replication and, thus, chromosome segregation. Moreover, increasing intracellular dCTP levels generates under-replication-induced sister-chromatid bridges as efficiently as PARP-1 knockdown. These results have direct implications for Bloom syndrome (BS), a rare genetic disease combining susceptibility to cancer and genomic instability. BS results from mutation of the BLM gene, encoding BLM, a RecQ 3′-5′ DNA helicase, a deficiency of which leads to CDA downregulation. BS cells thus have a CDA defect, resulting in a high frequency of ultrafine anaphase bridges due entirely to dCTP-dependent PARP-1 inhibition and independent of BLM status. Our study describes previously unknown pathological consequences of the distortion of dNTP pools and reveals an unexpected role for PARP-1 in preventing DNA under-replication and chromosome segregation defects.

Mohammed-Tayyib Boudra, Celeste Bolin, Sara Chiker, Alexis Fouquin, Tomasz Zaremba, Laurence Vaslin, Denis Biard, Fabrice P Cordelières, Frédérique Mégnin-Chanet, Vincent Favaudon, Marie Fernet, Vincent Pennaneach, Janet Hall (2015 Apr 1)

PARP-2 depletion results in lower radiation cell survival but cell line-specific differences in poly(ADP-ribose) levels.

Cellular and molecular life sciences : CMLS : 72 : 1585-1597 : DOI : 10.1007/s00018-014-1765-2 En savoir plus

Poly(ADP-ribose) polymerase-2 (PARP-2) activity contributes to a cells’ poly(ADP-ribosyl)ating potential and like PARP-1, has been implicated in several DNA repair pathways including base excision repair and DNA single strand break repair. Here the consequences of its stable depletion in HeLa, U20S, and AS3WT2 cells were examined. All three PARP-2 depleted models showed increased sensitivity to the cell killing effects on ionizing radiation as reported in PARP-2 depleted mouse embryonic fibroblasts providing further evidence for a role in DNA strand break repair. The PARP-2 depleted HeLa cells also showed both higher constitutive and DNA damage-induced levels of polymers of ADP-ribose (PAR) associated with unchanged PARP-1 protein levels, but higher PARP activity and a concomitant lower PARG protein levels and activity. These changes were accompanied by a reduced maximal recruitment of PARP-1, XRCC1, PCNA, and PARG to DNA damage sites. This PAR-associated phenotype could be reversed in HeLa cells on re-expression of PARP-2 and was not seen in U20S and AS3WT2 cells. These results highlight the complexity of the relationship between different members of the PARP family on PAR metabolism and suggest that cell model dependent phenotypes associated with the absence of PARP-2 exist within a common background of radiation sensitivity.


Année de publication : 2014

Martin Dutertre, Sarah Lambert, Aura Carreira, Mounira Amor-Guéret, Stéphan Vagner (2014 Mar 1)

DNA damage: RNA-binding proteins protect from near and far.

Trends in biochemical sciences : 141-9 : DOI : 10.1016/j.tibs.2014.01.003 En savoir plus

Recent work, including large-scale genetic and molecular analyses, identified RNA-binding proteins (RBPs) as major players in the prevention of genome instability. These studies show that RBPs prevent harmful RNA/DNA hybrids and are involved in the DNA damage response (DDR), from DNA repair to cell survival decisions. Indeed, specific RBPs allow the selective regulation of DDR genes at multiple post-transcriptional levels (from pre-mRNA splicing/polyadenylation to mRNA stability/translation) and are directly involved in DNA repair. These multiple activities are mediated by RBP binding to mRNAs, nascent transcripts, noncoding RNAs, and damaged DNA. Finally, because DNA damage modifies RBP localization and binding to different RNA/DNA molecules, we propose that upon DNA damage, RBPs coordinately regulate various aspects of both RNA and DNA metabolism.


Année de publication : 2012

Sébastien Rouzeau, Fabrice P Cordelières, Géraldine Buhagiar-Labarchède, Ilse Hurbain, Rosine Onclercq-Delic, Simon Gemble, Laura Magnaghi-Jaulin, Christian Jaulin, Mounira Amor-Guéret (2012 Apr 26)

Bloom’s syndrome and PICH helicases cooperate with topoisomerase IIα in centromere disjunction before anaphase.

PloS one : e33905 : DOI : 10.1371/journal.pone.0033905 En savoir plus

Centromeres are specialized chromosome domains that control chromosome segregation during mitosis, but little is known about the mechanisms underlying the maintenance of their integrity. Centromeric ultrafine anaphase bridges are physiological DNA structures thought to contain unresolved DNA catenations between the centromeres separating during anaphase. BLM and PICH helicases colocalize at these ultrafine anaphase bridges and promote their resolution. As PICH is detectable at centromeres from prometaphase onwards, we hypothesized that BLM might also be located at centromeres and that the two proteins might cooperate to resolve DNA catenations before the onset of anaphase. Using immunofluorescence analyses, we demonstrated the recruitment of BLM to centromeres from G2 phase to mitosis. With a combination of fluorescence in situ hybridization, electron microscopy, RNA interference, chromosome spreads and chromatin immunoprecipitation, we showed that both BLM-deficient and PICH-deficient prometaphase cells displayed changes in centromere structure. These cells also had a higher frequency of centromeric non disjunction in the absence of cohesin, suggesting the persistence of catenations. Both proteins were required for the correct recruitment to the centromere of active topoisomerase IIα, an enzyme specialized in the catenation/decatenation process. These observations reveal the existence of a functional relationship between BLM, PICH and topoisomerase IIα in the centromere decatenation process. They indicate that the higher frequency of centromeric ultrafine anaphase bridges in BLM-deficient cells and in cells treated with topoisomerase IIα inhibitors is probably due not only to unresolved physiological ultrafine anaphase bridges, but also to newly formed ultrafine anaphase bridges. We suggest that BLM and PICH cooperate in rendering centromeric catenates accessible to topoisomerase IIα, thereby facilitating correct centromere disjunction and preventing the formation of supernumerary centromeric ultrafine anaphase bridges.


Année de publication : 2011

Pauline Chabosseau, Géraldine Buhagiar-Labarchède, Rosine Onclercq-Delic, Sarah Lambert, Michelle Debatisse, Olivier Brison, Mounira Amor-Guéret (2011 Jun 28)

Pyrimidine pool imbalance induced by BLM helicase deficiency contributes to genetic instability in Bloom syndrome.

Nature communications : 368 : DOI : 10.1038/ncomms1363 En savoir plus

Defects in DNA replication are associated with genetic instability and cancer development, as illustrated in Bloom syndrome. Features of this syndrome include a slowdown in replication speed, defective fork reactivation and high rates of sister chromatid exchange, with a general predisposition to cancer. Bloom syndrome is caused by mutations in the BLM gene encoding a RecQ helicase. Here we report that BLM deficiency is associated with a strong cytidine deaminase defect, leading to pyrimidine pool disequilibrium. In BLM-deficient cells, pyrimidine pool normalization leads to reduction of sister chromatid exchange frequency and is sufficient for full restoration of replication fork velocity but not the fork restart defect, thus identifying the part of the Bloom syndrome phenotype because of pyrimidine pool imbalance. This study provides new insights into the molecular basis of control of replication speed and the genetic instability associated with Bloom syndrome. Nucleotide pool disequilibrium could be a general phenomenon in a large spectrum of precancerous and cancer cells.