UMR3348 – Genome integrity, RNA and Cancer

Unit publications

Year of publication 2021

Åsa Ehlén, Gaetana Sessa, Sophie Zinn-Justin, Aura Carreira (2021 Mar 10)

The phospho-dependent role of BRCA2 on the maintenance of chromosome integrity.

Cell cycle (Georgetown, Tex.) : 1-12 : DOI : 10.1080/15384101.2021.1892994 Learn more
Summary

Chromosomal instability is a hallmark of cancer. The tumor suppressor protein BRCA2 performs an important role in the maintenance of genome integrity particularly in interphase; as a mediator of homologous recombination DNA repair pathway, it participates in the repair of DNA double-strand breaks, inter-strand crosslinks and replicative DNA lesions. BRCA2 also protects stalled replication forks from aberrant degradation. Defects in these functions lead to structural chromosomal aberrations. BRCA2 is a large protein containing highly disordered regions that are heavily phosphorylated particularly in mitosis. The functions of these modifications are getting elucidated and reveal emerging activities in chromosome alignment, chromosome segregation and abscission during cell division. Defects in these activities result in numerical chromosomal aberrations. In addition to BRCA2, other factors of the DNA damage response (DDR) participate in mitosis in close association with cell cycle kinases and phosphatases suggesting that the maintenance of genome integrity functions of these factors extends beyond DNA repair. Here we will discuss the regulation of BRCA2 functions through phosphorylation by cell cycle kinases particularly in mitosis, and illustrate with some examples how BRCA2 and other DDR proteins partially rewire their interactions, essentially via phosphorylation, to fulfill mitotic specific functions that ensure chromosome stability.

Fold up
Martin Dutertre, Rym Sfaxi, Stéphan Vagner (2021 Feb 27)

Reciprocal Links between Pre-messenger RNA 3′-End Processing and Genome Stability.

Trends in biochemical sciences : DOI : S0968-0004(21)00023-2 Learn more
Summary

The 3′-end processing of most pre-messenger RNAs (pre-mRNAs) involves RNA cleavage and polyadenylation and is coupled to transcription termination. In both yeast and human cells, pre-mRNA 3′-end cleavage is globally inhibited by DNA damage. Recently, further links between pre-mRNA 3′-end processing and the control of genome stability have been uncovered, as reviewed here. Upon DNA damage, various genes related to the DNA damage response (DDR) escape 3′-end processing inhibition or are regulated through alternative polyadenylation (APA). Conversely, various pre-mRNA 3′-end processing factors prevent genome instability and are found at sites of DNA damage. Finally, the reciprocal link between pre-mRNA 3′-end processing and genome stability control seems important because it is conserved in evolution and involved in disease development.

Fold up
Gaetana Sessa, Belén Gómez-González, Sonia Silva, Carmen Pérez-Calero, Romane Beaurepere, Sonia Barroso, Sylvain Martineau, Charlotte Martin, Åsa Ehlén, Juan S Martínez, Bérangère Lombard, Damarys Loew, Stephan Vagner, Andrés Aguilera, Aura Carreira (2021 Feb 26)

BRCA2 promotes R-loop resolution by DDX5 helicase at DNA breaks to facilitate their repair by homologous recombination.

The EMBO journal : e106018 : DOI : 10.15252/embj.2020106018 Learn more
Summary

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.

Fold up
Satish Bodakuntla, Carsten Janke, Maria M Magiera (2021 Jan 19)

Tubulin polyglutamylation, a regulator of microtubule functions, can cause neurodegeneration.

Neuroscience letters : 135656 : DOI : S0304-3940(21)00034-3 Learn more
Summary

Neurodegenerative diseases lead to a progressive demise of neuronal functions that ultimately results in neuronal death. Besides a large variety of molecular pathways that have been linked to the degeneration of neurons, dysfunctions of the microtubule cytoskeleton are common features of many human neurodegenerative disorders. Yet, it is unclear whether microtubule dysfunctions are causative, or mere bystanders in the disease progression. A so-far little explored regulatory mechanism of the microtubule cytoskeleton, the posttranslational modifications of tubulin, emerge as candidate mechanisms involved in neuronal dysfunction, and thus, degeneration. Here we review the role of tubulin polyglutamylation, a prominent modification of neuronal microtubules. We discuss the current understanding of how polyglutamylation controls microtubule functions in healthy neurons, and how deregulation of this modification leads to neurodegeneration in mice and humans.

Fold up
Sudarshan Gadadhar, Gonzalo Alvarez Viar, Jan Niklas Hansen, An Gong, Aleksandr Kostarev, Côme Ialy-Radio, Sophie Leboucher, Marjorie Whitfield, Ahmed Ziyyat, Aminata Touré, Luis Alvarez, Gaia Pigino , Carsten Janke (2021 Jan 8)

Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility

ScienceScience : DOI : 10.1126/science.abd4914 Learn more
Summary

Abstract

Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo-electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility.

Fold up

Year of publication 2020

Shensi Shen, Stéphan Vagner, Caroline Robert (2020 Nov 12)

Persistent Cancer Cells: The Deadly Survivors.

Cell : 860-874 : DOI : S0092-8674(20)31391-X Learn more
Summary

Persistent cancer cells are the discrete and usually undetected cells that survive cancer drug treatment and constitute a major cause of treatment failure. These cells are characterized by their slow proliferation, highly flexible energy consumption, adaptation to their microenvironment, and phenotypic plasticity. Mechanisms that underlie their persistence offer highly coveted and sought-after therapeutic targets, and include diverse epigenetic, transcriptional, and translational regulatory processes, as well as complex cell-cell interactions. Although the successful clinical targeting of persistent cancer cells remains to be realized, immense progress has been made in understanding their persistence, yielding promising preclinical results.

Fold up
Karol Kramarz, Kamila Schirmeisen, Virginie Boucherit, Anissia Ait Saada, Claire Lovo, Benoit Palancade, Catherine Freudenreich, Sarah A E Lambert (2020 Nov 7)

The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal.

Nature communications : 5643 : DOI : 10.1038/s41467-020-19516-z Learn more
Summary

Nuclear Pore complexes (NPCs) act as docking sites to anchor particular DNA lesions facilitating DNA repair by elusive mechanisms. Using replication fork barriers in fission yeast, we report that relocation of arrested forks to NPCs occurred after Rad51 loading and its enzymatic activity. The E3 SUMO ligase Pli1 acts at arrested forks to safeguard integrity of nascent strands and generates poly-SUMOylation which promote relocation to NPCs but impede the resumption of DNA synthesis by homologous recombination (HR). Anchorage to NPCs allows SUMO removal by the SENP SUMO protease Ulp1 and the proteasome, promoting timely resumption of DNA synthesis. Preventing Pli1-mediated SUMO chains was sufficient to bypass the need for anchorage to NPCs and the inhibitory effect of poly-SUMOylation on HR-mediated DNA synthesis. Our work establishes a novel spatial control of Recombination-Dependent Replication (RDR) at a unique sequence that is distinct from mechanisms engaged at collapsed-forks and breaks within repeated sequences.

https://www-nature-com.insb.bib.cnrs.fr/articles/s41467-020-19516-z

Fold up
Satish Bodakuntla, A S Jijumon, Carsten Janke, Maria M Magiera (2020 Nov 5)

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles.

Journal of visualized experiments : JoVE : DOI : 10.3791/61826 Learn more
Summary

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The “tubulin code” is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs. To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps. We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.

Fold up
Shubham Kesarwani, Prakash Lama, Anchal Chandra, P Purushotam Reddy, A S Jijumon, Satish Bodakuntla, Balaji M Rao, Carsten Janke, Ranabir Das, Minhajuddin Sirajuddin (2020 Oct 5)

Genetically encoded live-cell sensor for tyrosinated microtubules.

The Journal of cell biology : DOI : e201912107 Learn more
Summary

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells.

Fold up
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 Learn more
Summary

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.

Fold up
L Boeckemeier, R Kraehenbuehl, A Keszthelyi, M U Gasasira, E G Vernon, R Beardmore, C B Vågbø, D Chaplin, S Gollins, H E Krokan, S A E Lambert, B Paizs, E Hartsuiker (2020 May 29)

Mre11 exonuclease activity removes the chain-terminating nucleoside analog gemcitabine from the nascent strand during DNA replication.

Science advances : eaaz4126 : DOI : 10.1126/sciadv.aaz4126 Learn more
Summary

The Mre11 nuclease is involved in early responses to DNA damage, often mediated by its role in DNA end processing. mutations and aberrant expression are associated with carcinogenesis and cancer treatment outcomes. While, in recent years, progress has been made in understanding the role of Mre11 nuclease activities in DNA double-strand break repair, their role during replication has remained elusive. The nucleoside analog gemcitabine, widely used in cancer therapy, acts as a replication chain terminator; for a cell to survive treatment, gemcitabine needs to be removed from replicating DNA. Activities responsible for this removal have, so far, not been identified. We show that Mre11 3′ to 5′ exonuclease activity removes gemcitabine from nascent DNA during replication. This contributes to replication progression and gemcitabine resistance. We thus uncovered a replication-supporting role for Mre11 exonuclease activity, which is distinct from its previously reported detrimental role in uncontrolled resection in recombination-deficient cells.

Fold up
Iris Tanaka, Alina Chakraborty, Olivier Saulnier, Clara Benoit-Pilven, Sophie Vacher, Dalila Labiod, Eric W F Lam, Ivan Bièche, Olivier Delattre, Frédéric Pouzoulet, Didier Auboeuf, Stéphan Vagner, Martin Dutertre (2020 Jan 17)

ZRANB2 and SYF2-mediated splicing programs converging on ECT2 are involved in breast cancer cell resistance to doxorubicin.

Nucleic acids research : DOI : gkz1213 Learn more
Summary

Besides analyses of specific alternative splicing (AS) variants, little is known about AS regulatory pathways and programs involved in anticancer drug resistance. Doxorubicin is widely used in breast cancer chemotherapy. Here, we identified 1723 AS events and 41 splicing factors regulated in a breast cancer cell model of acquired resistance to doxorubicin. An RNAi screen on splicing factors identified the little studied ZRANB2 and SYF2, whose depletion partially reversed doxorubicin resistance. By RNAi and RNA-seq in resistant cells, we found that the AS programs controlled by ZRANB2 and SYF2 were enriched in resistance-associated AS events, and converged on the ECT2 splice variant including exon 5 (ECT2-Ex5+). Both ZRANB2 and SYF2 were found associated with ECT2 pre-messenger RNA, and ECT2-Ex5+ isoform depletion reduced doxorubicin resistance. Following doxorubicin treatment, resistant cells accumulated in S phase, which partially depended on ZRANB2, SYF2 and the ECT2-Ex5+ isoform. Finally, doxorubicin combination with an oligonucleotide inhibiting ECT2-Ex5 inclusion reduced doxorubicin-resistant tumor growth in mouse xenografts, and high ECT2-Ex5 inclusion levels were associated with bad prognosis in breast cancer treated with chemotherapy. Altogether, our data identify AS programs controlled by ZRANB2 and SYF2 and converging on ECT2, that participate to breast cancer cell resistance to doxorubicin.

Fold up

Year of publication 2019

Julien Hardy, Dingli Dai, Anissia Ait Saada, Ana Teixeira-Silva, Louise Dupoiron, Fatemeh Mojallali, Karine Fréon, Francoise Ochsenbein, Brigitte Hartmann, Sarah Lambert (2019 Oct 4)

Histone deposition promotes recombination-dependent replication at arrested forks.

PLoS genetics : e1008441 : DOI : 10.1371/journal.pgen.1008441 Learn more
Summary

Replication stress poses a serious threat to genome stability. Recombination-Dependent-Replication (RDR) promotes DNA synthesis resumption from arrested forks. Despite the identification of chromatin restoration pathways after DNA repair, crosstalk coupling RDR and chromatin assembly is largely unexplored. The fission yeast Chromatin Assembly Factor-1, CAF-1, is known to promote RDR. Here, we addressed the contribution of histone deposition to RDR. We expressed a mutated histone, H3-H113D, to genetically alter replication-dependent chromatin assembly by destabilizing (H3-H4)2 tetramer. We established that DNA synthesis-dependent histone deposition, by CAF-1 and Asf1, promotes RDR by preventing Rqh1-mediated disassembly of joint-molecules. The recombination factor Rad52 promotes CAF-1 binding to sites of recombination-dependent DNA synthesis, indicating that histone deposition occurs downstream Rad52. Histone deposition and Rqh1 activity act synergistically to promote cell resistance to camptothecin, a topoisomerase I inhibitor that induces replication stress. Moreover, histone deposition favors non conservative recombination events occurring spontaneously in the absence of Rqh1, indicating that the stabilization of joint-molecules by histone deposition also occurs independently of Rqh1 activity. These results indicate that histone deposition plays an active role in promoting RDR, a benefit counterbalanced by stabilizing at-risk joint-molecules for genome stability.

Fold up
Anissia Ait-Saada, Olga Khorosjutina, Jiang Chen, Karol Kramarz, Vladimir Maksimov, J Peter Svensson, Sarah Lambert, Karl Ekwall (2019 Oct 1)

Chromatin remodeler Fft3 plays a dual role at blocked DNA replication forks.

Life science alliance : DOI : e201900433 Learn more
Summary

Here, we investigate the function of fission yeast Fun30/Smarcad1 family of SNF2 ATPase-dependent chromatin remodeling enzymes in DNA damage repair. There are three Fun30 homologues in fission yeast, Fft1, Fft2, and Fft3. We find that only Fft3 has a function in DNA repair and it is needed for single-strand annealing of an induced double-strand break. Furthermore, we use an inducible replication fork barrier system to show that Fft3 has two distinct roles at blocked DNA replication forks. First, Fft3 is needed for the resection of nascent strands, and second, it is required to restart the blocked forks. The latter function is independent of its ATPase activity.

Fold up
Judith Souphron, Satish Bodakuntla, A S Jijumon, Goran Lakisic, Alexis M Gautreau, Carsten Janke, Maria M Magiera (2019 Apr 19)

Purification of tubulin with controlled post-translational modifications by polymerization-depolymerization cycles.

Nature protocols : DOI : 10.1038/s41596-019-0153-7 Learn more
Summary

In vitro reconstitutions of microtubule assemblies have provided essential mechanistic insights into the molecular bases of microtubule dynamics and their interactions with associated proteins. The tubulin code has emerged as a regulatory mechanism for microtubule functions, which suggests that tubulin isotypes and post-translational modifications (PTMs) play important roles in controlling microtubule functions. To investigate the tubulin code mechanism, it is essential to analyze different tubulin variants in vitro. Until now, this has been difficult, as most reconstitution experiments have used heavily post-translationally modified tubulin purified from brain tissue. Therefore, we developed a protocol that allows purification of tubulin with controlled PTMs from limited sources through cycles of polymerization and depolymerization. Although alternative protocols using affinity purification of tubulin also yield very pure tubulin, our protocol has the unique advantage of selecting for fully functional tubulin, as non-polymerizable tubulin is excluded in the successive polymerization cycles. It thus provides a novel procedure for obtaining tubulin with controlled PTMs for in vitro reconstitution experiments. We describe specific procedures for tubulin purification from adherent cells, cells grown in suspension cultures and single mouse brains. The protocol can be combined with drug treatment, transfection of cells before tubulin purification or enzymatic treatment during the purification process. The amplification of cells and their growth in spinner bottles takes ~13 d; the tubulin purification takes 6-7 h. The tubulin can be used in total internal reflection fluorescence (TIRF)-microscopy-based experiments or pelleting assays for the investigation of intrinsic properties of microtubules and their interactions with associated proteins.

Fold up