UMR3348 – Intégrité du génome, ARN et cancer

Publications de l’unité

Année de publication : 2020

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 En savoir plus
Résumé

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.

Replier
Yanzhang Luo, ShengQi Xiang, Peter Jan Hooikaas, Laura van Bezouwen, A S Jijumon, Carsten Janke, Friedrich Förster, Anna Akhmanova, Marc Baldus (2020 Jan 2)

Direct observation of dynamic protein interactions involving human microtubules using solid-state NMR spectroscopy.

Nature communications : 18 : DOI : 10.1038/s41467-019-13876-x En savoir plus
Résumé

Microtubules are important components of the eukaryotic cytoskeleton. Their structural organization is regulated by nucleotide binding and many microtubule-associated proteins (MAPs). While cryo-EM and X-ray crystallography have provided detailed views of interactions between MAPs with the microtubule lattice, little is known about how MAPs and their intrinsically disordered regions interact with the dynamic microtubule surface. NMR carries the potential to directly probe such interactions but so far has been precluded by the low tubulin yield. We present a protocol to produce [C, N]-labeled, functional microtubules (MTs) from human cells for solid-state NMR studies. This approach allowed us to demonstrate that MAPs can differently modulate the fast time-scale dynamics of C-terminal tubulin tails, suggesting distinct interaction modes. Our results pave the way for in-depth NMR studies of protein dynamics involved in MT assembly and their interactions with other cellular components.

Replier

Année de publication : 2019

Shensi Shen, Sara Faouzi, Amandine Bastide, Sylvain Martineau, Hélène Malka-Mahieu, Yu Fu, Xiaoxiao Sun, Christine Mateus, Emilie Routier, Severine Roy, Laurent Desaubry, Fabrice André, Alexander Eggermont, Alexandre David, Jean-Yves Scoazec, Stéphan Vagner, Caroline Robert (2019 Dec 18)

An epitranscriptomic mechanism underlies selective mRNA translation remodelling in melanoma persister cells.

Nature communications : 5713 : DOI : 10.1038/s41467-019-13360-6 En savoir plus
Résumé

Cancer persister cells tolerate anticancer drugs and serve as the founders of acquired resistance and cancer relapse. Here we show that a subpopulation of BRAF mutant melanoma cells that tolerates exposure to BRAF and MEK inhibitors undergoes a reversible remodelling of mRNA translation that evolves in parallel with drug sensitivity. Although this process is associated with a global reduction in protein synthesis, a subset of mRNAs undergoes an increased efficiency in translation. Inhibiting the eIF4A RNA helicase, a component of the eIF4F translation initiation complex, abrogates this selectively increased translation and is lethal to persister cells. Translation remodelling in persister cells coincides with an increased N6-methyladenosine modification in the 5′-untranslated region of some highly translated mRNAs. Combination of eIF4A inhibitor with BRAF and MEK inhibitors effectively inhibits the emergence of persister cells and may represent a new therapeutic strategy to prevent acquired drug resistance.

Replier
Aviel Even, Giovanni Morelli, Loïc Broix, Chiara Scaramuzzino, Silvia Turchetto, Ivan Gladwyn-Ng, Romain Le Bail, Michal Shilian, Stephen Freeman, Maria M Magiera, A S Jijumon, Nathalie Krusy, Brigitte Malgrange, Bert Brone, Paula Dietrich, Ioannis Dragatsis, Carsten Janke, Frédéric Saudou, Miguel Weil, Laurent Nguyen (2019 Dec 18)

ATAT1-enriched vesicles promote microtubule acetylation via axonal transport.

Science advances : eaax2705 : DOI : 10.1126/sciadv.aax2705 En savoir plus
Résumé

Microtubules are polymerized dimers of α- and β-tubulin that underlie a broad range of cellular activities. Acetylation of α-tubulin by the acetyltransferase ATAT1 modulates microtubule dynamics and functions in neurons. However, it remains unclear how this enzyme acetylates microtubules over long distances in axons. Here, we show that loss of ATAT1 impairs axonal transport in neurons in vivo, and cell-free motility assays confirm a requirement of α-tubulin acetylation for proper bidirectional vesicular transport. Moreover, we demonstrate that the main cellular pool of ATAT1 is transported at the cytosolic side of neuronal vesicles that are moving along axons. Together, our data suggest that axonal transport of ATAT1-enriched vesicles is the predominant driver of α-tubulin acetylation in axons.

Replier
Satish Bodakuntla, A S Jijumon, Cristopher Villablanca, Christian Gonzalez-Billault, Carsten Janke (2019 Oct 29)

Microtubule-Associated Proteins: Structuring the Cytoskeleton.

Trends in cell biology : 804-819 : DOI : S0962-8924(19)30120-5 En savoir plus
Résumé

Microtubule-associated proteins (MAPs) were initially discovered as proteins that bind to and stabilize microtubules. Today, an ever-growing number of MAPs reveals a more complex picture of these proteins as organizers of the microtubule cytoskeleton that have a large variety of functions. MAPs enable microtubules to participate in a plethora of cellular processes such as the assembly of mitotic and meiotic spindles, neuronal development, and the formation of the ciliary axoneme. Although some subgroups of MAPs have been exhaustively characterized, a strikingly large number of MAPs remain barely characterized other than their interactions with microtubules. We provide a comprehensive view on the currently known MAPs in mammals. We discuss their molecular mechanisms and functions, as well as their physiological role and links to pathologies.

Replier
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 En savoir plus
Résumé

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.

Replier
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 En savoir plus
Résumé

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.

Replier
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 En savoir plus
Résumé

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.

Replier
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
Résumé

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.

Replier
Sarah Lambert (2019 Mar 3)

Unstable genomes promote inflammation.

Nature : 41-42 : DOI : 10.1038/d41586-019-00510-5 En savoir plus
Résumé

Replier
Catherine Strassel, Maria M Magiera, Arnaud Dupuis, Morgane Batzenschlager, Agnès Hovasse, Irina Pleines, Paul Guéguen, Anita Eckly, Sylvie Moog, Léa Mallo, Quentin Kimmerlin, Stéphane Chappaz, Jean-Marc Strub, Natarajan Kathiresan, Henri de la Salle, Alain Van Dorsselaer, Claude Ferec, Jean-Yves Py, Christian Gachet, Christine Schaeffer-Reiss, Benjamin T Kile, Carsten Janke, François Lanza (2019 Feb 15)

An essential role for α4A-tubulin in platelet biogenesis.

Life science alliance : DOI : e201900309 En savoir plus
Résumé

During platelet biogenesis, microtubules (MTs) are arranged into submembranous structures (the marginal band) that encircle the cell in a single plane. This unique MT array has no equivalent in any other mammalian cell, and the mechanisms responsible for this particular mode of assembly are not fully understood. One possibility is that platelet MTs are composed of a particular set of tubulin isotypes that carry specific posttranslational modifications. Although β1-tubulin is known to be essential, no equivalent roles of α-tubulin isotypes in platelet formation or function have so far been reported. Here, we identify α4A-tubulin as a predominant α-tubulin isotype in platelets. Similar to β1-tubulin, α4A-tubulin expression is up-regulated during the late stages of megakaryocyte differentiation. Missense mutations in the α4A-tubulin gene cause macrothrombocytopenia in mice and humans. Defects in α4A-tubulin lead to changes in tubulin tyrosination status of the platelet tubulin pool. Ultrastructural defects include reduced numbers and misarranged MT coils in the platelet marginal band. We further observed defects in megakaryocyte maturation and proplatelet formation in -mutant mice. We have, thus, discovered an α-tubulin isotype with specific and essential roles in platelet biogenesis.

Replier
Tiziana Giordano, Sudarshan Gadadhar, Satish Bodakuntla, Jonas Straub, Sophie Leboucher, Guillaume Martinez, Walid Chemlali, Christophe Bosc, Annie Andrieux, Ivan Bieche, Christophe Arnoult, Stefan Geimer, Carsten Janke (2019 Feb 7)

Loss of the deglutamylase CCP5 perturbs multiple steps of spermatogenesis and leads to male infertility.

Journal of cell science : DOI : jcs226951 En savoir plus
Résumé

Sperm cells are highly specialized mammalian cells, and their biogenesis requires unique intracellular structures. Perturbation of spermatogenesis often leads to male infertility. Here, we assess the role of a post-translational modification of tubulin, glutamylation, in spermatogenesis. We show that mice lacking the tubulin deglutamylase CCP5 (also known as AGBL5) do not form functional sperm. In these mice, spermatids accumulate polyglutamylated tubulin, accompanied by the occurrence of disorganized microtubule arrays, in particular in the sperm manchette. Spermatids further fail to re-arrange their intracellular space and accumulate organelles and cytosol, while nuclei condense normally. Strikingly, spermatids lacking CCP5 show supernumerary centrioles, suggesting that glutamylation could control centriole duplication. We show that most of these observed defects are also present in mice in which CCP5 is deleted only in the male germ line, strongly suggesting that they are germ-cell autonomous. Our findings reveal that polyglutamylation is, beyond its known importance for sperm flagella, an essential regulator of several microtubule-based functions during spermatogenesis. This makes enzymes involved in glutamylation prime candidates for being genes involved in male sterility.

Replier
Pedro Guedes-Dias, Jeffrey J Nirschl, Nohely Abreu, Mariko K Tokito, Carsten Janke, Maria M Magiera, Erika L F Holzbaur (2019 Jan 21)

Kinesin-3 Responds to Local Microtubule Dynamics to Target Synaptic Cargo Delivery to the Presynapse.

Current biology : CB : 268-282.e8 : DOI : S0960-9822(18)31595-1 En savoir plus
Résumé

Neurons in the CNS establish thousands of en passant synapses along their axons. Robust neurotransmission depends on the replenishment of synaptic components in a spatially precise manner. Using live-cell microscopy and single-molecule reconstitution assays, we find that the delivery of synaptic vesicle precursors (SVPs) to en passant synapses in hippocampal neurons is specified by an interplay between the kinesin-3 KIF1A motor and presynaptic microtubules. Presynaptic sites are hotspots of dynamic microtubules rich in GTP-tubulin. KIF1A binds more weakly to GTP-tubulin than GDP-tubulin and competes with end-binding (EB) proteins for binding to the microtubule plus end. A disease-causing mutation within KIF1A that reduces preferential binding to GDP- versus GTP-rich microtubules disrupts SVP delivery and reduces presynaptic release upon neuronal stimulation. Thus, the localized enrichment of dynamic microtubules along the axon specifies a localized unloading zone that ensures the accurate delivery of SVPs, controlling presynaptic strength in hippocampal neurons.

Replier
Hannah L Klein, Giedrė Bačinskaja, Jun Che, Anais Cheblal, Rajula Elango, Anastasiya Epshtein, Devon M Fitzgerald, Belén Gómez-González, Sharik R Khan, Sandeep Kumar, Bryan A Leland, Léa Marie, Qian Mei, Judith Miné-Hattab, Alicja Piotrowska, Erica J Polleys, Christopher D Putnam, Elina A Radchenko, Anissia Ait Saada, Cynthia J Sakofsky, Eun Yong Shim, Mathew Stracy, Jun Xia, Zhenxin Yan, Yi Yin, Andrés Aguilera, Juan Lucas Argueso, Catherine H Freudenreich, Susan M Gasser, Dmitry A Gordenin, James E Haber, Grzegorz Ira, Sue Jinks-Robertson, Megan C King, Richard D Kolodner, Andrei Kuzminov, Sarah Ae Lambert, Sang Eun Lee, Kyle M Miller, Sergei M Mirkin, Thomas D Petes, Susan M Rosenberg, Rodney Rothstein, Lorraine S Symington, Pawel Zawadzki, Nayun Kim, Michael Lisby, Anna Malkova (2019 Jan 7)

Guidelines for DNA recombination and repair studies: Cellular assays of DNA repair pathways.

Microbial cell (Graz, Austria) : 1-64 : DOI : 10.15698/mic2019.01.664 En savoir plus
Résumé

Understanding the plasticity of genomes has been greatly aided by assays for recombination, repair and mutagenesis. These assays have been developed in microbial systems that provide the advantages of genetic and molecular reporters that can readily be manipulated. Cellular assays comprise genetic, molecular, and cytological reporters. The assays are powerful tools but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

Replier

Année de publication : 2018

Athanasia Stoupa, Frédéric Adam, Dulanjalee Kariyawasam, Catherine Strassel, Sanjay Gawade, Gabor Szinnai, Alexandre Kauskot, Dominique Lasne, Carsten Janke, Kathiresan Natarajan, Alain Schmitt, Christine Bole-Feysot, Patrick Nitschke, Juliane Léger, Fabienne Jabot-Hanin, Frédéric Tores, Anita Michel, Arnold Munnich, Claude Besmond, Raphaël Scharfmann, François Lanza, Delphine Borgel, Michel Polak, Aurore Carré (2018 Nov 18)

TUBB1 mutations cause thyroid dysgenesis associated with abnormal platelet physiology.

EMBO molecular medicine : DOI : e9569 En savoir plus
Résumé

The genetic causes of congenital hypothyroidism due to thyroid dysgenesis (TD) remain largely unknown. We identified three novel gene mutations that co-segregated with TD in three distinct families leading to 1.1% of mutations in TD study cohort. (Tubulin, Beta 1 Class VI) encodes for a member of the β-tubulin protein family. gene is expressed in the developing and adult thyroid in humans and mice. All three mutations lead to non-functional α/β-tubulin dimers that cannot be incorporated into microtubules. In mice, knock-out disrupted microtubule integrity by preventing β1-tubulin incorporation and impaired thyroid migration and thyroid hormone secretion. In addition, mutations caused the formation of macroplatelets and hyperaggregation of human platelets after stimulation by low doses of agonists. Our data highlight unexpected roles for β1-tubulin in thyroid development and in platelet physiology. Finally, these findings expand the spectrum of the rare paediatric diseases related to mutations in tubulin-coding genes and provide new insights into the genetic background and mechanisms involved in congenital hypothyroidism and thyroid dysgenesis.

Replier