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1. SHLD1 is dispensable for 53BP1-dependent V(D)J recombination but critical for productive class switch recombination

Author

Estelle Vincendeau, Wenming Wei, Xuefei Zhang, Cyril Planchais, Wei Yu, Hélène Lenden-Hasse, Thomas Cokelaer, Juliana Pipoli da Fonseca, Hugo Mouquet, David J. Adams, Frederick W. Alt, Stephen P. Jackson, Gabriel Balmus, Chloé Lescale, Ludovic Deriano, Vincendeau, Estelle [0000-0002-0231-1609], Zhang, Xuefei [0000-0002-1873-6679], Yu, Wei [0000-0002-1777-5254], Cokelaer, Thomas [0000-0001-6286-1138], Adams, David J [0000-0001-9490-0306], Jackson, Stephen P [0000-0001-9317-7937], Balmus, Gabriel [0000-0003-2872-4468], Lescale, Chloé [0000-0003-0622-1149], Deriano, Ludovic [0000-0002-9673-9525], Apollo - University of Cambridge Repository, Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Howard Hughes Medical Institute (HHMI), Peking University [Beijing], Immunologie humorale - Humoral Immunology, Biomics (plateforme technologique), Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, The Wellcome Trust Sanger Institute [Cambridge], University of Cambridge [UK] (CAM), The Deriano laboratory is funded by Institut Pasteur, Institut National de la Santé et de la Recherche Médicale (Inserm), Wordwide Cancer Research (grant # 19-0333), Ligue Nationale Contre le Cancer (Equipe labellisée 2019), and Institut National du Cancer (INCa, grant # PLBIO19-122). E.V. is supported by the French Ministry of Higher Education, Research, and Innovation (Doctoral School Bio Sorbonne Paris Cité) and by the Fondation pour la Recherche Médicale. W.W. is part of the Pasteur–Paris University International Ph.D. program and received funding from the CNBG company, China. The Balmus laboratory at the UK DRI is funded by the Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK. Research in the Jackson laboratory is funded by Cancer Research UK (CRUK) program grant DRCPGM\100005 and core grant C6946/A24843, Wellcome Investigator award 206388/Z/17/Z and core grant 203144, and ERC Synergy award 855741. S.P.J. receives a salary from the University of Cambridge. F.W.A is an investigator of the Howard Hughes Medical Institute. H.M. received core grants from the Institut Pasteur, the INSERM and the Milieu Intérieur Program (ANR-10-LABX-69-01)., We thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders for providing the mutant mouse line (Allele: Shld1em1(IMPC)Wtsi), and INFRAFRONTIER/EMMA. We thank the Institut Pasteur animal facility, specifically Quentin Mille, Jeromine Rohard Blanco, and Franck Bourgade for mouse weighing. We thank Jean-Pierre de Villartay for providing the CD21-Cre3a line and Klaus Rajewsky and MGC for permission to use the line. We thank Michela Di Virgilio for providing 53BP1 knockout animals and Junjie Chen for permission to use them. We thank members of the Deriano lab for their discussion and technical help. We thank Bernardo Reina-San-Martin for the discussion and advices. We thank Christopher Carnie for the discussion on the design of SHLD1 mutant sites., ANR-10-LABX-0069,MILIEU INTERIEUR,GENETIC & ENVIRONMENTAL CONTROL OF IMMUNE PHENOTYPE VARIANCE: ESTABLISHING A PATH TOWARDS PERSONALIZED MEDICINE(2010), and Jackson, Stephen [0000-0001-9317-7937]

Subjects
DNA End-Joining Repair, MESH: V(D)J Recombination, [SDV]Life Sciences [q-bio], MESH: DNA Breaks, Double-Stranded, General Physics and Astronomy, 13/106, 45/23, 631/250/2152/2497, 631/250/2152/2498, General Biochemistry, Genetics and Molecular Biology, 13/1, 13/21, DNA Breaks, Double-Stranded, 631/337/1427/2122, Multidisciplinary, 45/71, 45, fungi, article, 45/77, General Chemistry, MESH: DNA End-Joining Repair, Immunoglobulin Class Switching, V(D)J Recombination, 49, DNA-Binding Proteins, 13/31, enzymes and coenzymes (carbohydrates), MESH: Immunoglobulin Class Switching, 631/337/149, MESH: DNA-Binding Proteins
Abstract

Funder: Ligue Nationale Contre le Cancer, SHLD1 is part of the Shieldin (SHLD) complex, which acts downstream of 53BP1 to counteract DNA double-strand break (DSB) end resection and promote DNA repair via non-homologous end-joining (NHEJ). While 53BP1 is essential for immunoglobulin heavy chain class switch recombination (CSR), long-range V(D)J recombination and repair of RAG-induced DSBs in XLF-deficient cells, the function of SHLD during these processes remains elusive. Here we report that SHLD1 is dispensable for lymphocyte development and RAG-mediated V(D)J recombination, even in the absence of XLF. By contrast, SHLD1 is essential for restricting resection at AID-induced DSB ends in both NHEJ-proficient and NHEJ-deficient B cells, providing an end-protection mechanism that permits productive CSR by NHEJ and alternative end-joining. Finally, we show that this SHLD1 function is required for orientation-specific joining of AID-initiated DSBs. Our data thus suggest that 53BP1 promotes V(D)J recombination and CSR through two distinct mechanisms: SHLD-independent synapsis of V(D)J segments and switch regions within chromatin, and SHLD-dependent protection of AID-DSB ends against resection.

Published
2022
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2. Silver Russell syndrome in a preterm girl with 8q12.1 deletion encompassing PLAG1

Author

I K Temple, Sandra Chantot-Bastaraud, Jose María Lloreda-García, Maria Olmo-Sanchez, Cristina De la Torre-Sandoval, Jose Ramón Fernández-Fructuoso, Madeleine D. Harbison, Irene Netchine, Couvet, Sandrine, Hospital General Universitario Santa Lucía (Cartagena), Icahn School of Medicine at Mount Sinai [New York] (MSSM), Maladies génétiques d'expression pédiatrique [CHU Trousseau] (Inserm U933), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), UF de Génétique chromosomique [CHU Trousseau], CHU Trousseau [APHP], University of Southampton, Centre de Recherche Saint-Antoine (CRSA), and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)

Subjects
Pathology, medicine.medical_specialty, [SDV]Life Sciences [q-bio], media_common.quotation_subject, medicine.medical_treatment, [SDV.GEN.GH] Life Sciences [q-bio]/Genetics/Human genetics, MESH: Phenotype, Pathology and Forensic Medicine, Feeding difficulty, parasitic diseases, medicine, Humans, Girl, Genetics (clinical), media_common, Gynecology, MESH: Humans, Growth retardation, business.industry, Growth factor, Silver–Russell syndrome, Postnatal growth retardation, MESH: Transcription Factors, General Medicine, medicine.disease, Phenotype, DNA-Binding Proteins, [SDV] Life Sciences [q-bio], Silver-Russell Syndrome, MESH: Silver-Russell Syndrome, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Pediatrics, Perinatology and Child Health, Female, Anatomy, Haploinsufficiency, business, MESH: Female, MESH: DNA-Binding Proteins, Transcription Factors, Congenital disorder
Abstract

Silver Russell syndrome (SRS) is a congenital disorder characterised by intrauterine growth retardation (IUGR), feeding difficulties and postnatal growth retardation. In a small number of cases PLAG1 variants have been described (OMIM #618907). PLAG1 haploinsufficiency decreases IGF2 expression and produces a Silver Russell syndrome like phenotype. Here, we describe the phenotype and molecular features of a 26 months girl with clinical features of SRS and a de novo 2.1 Mb deletion encompassing PLAG1 is reported in association with clinical features suggestive of SRS. This manuscript has been published in "Clinical Dysmorphology". https://journals.lww.com/clindysmorphol/Abstract/2021/10000/Silver_Russell_syndrome_in_a_preterm_girl_with.7.aspxMust be cited as follows: Fernandez-Fructuoso JR, De la Torre-Sandoval C, Harbison MD, Chantot-Bastaraud S, Temple K, Lloreda-Garcia JM, Olmo-Sanchez M, Netchine I. Silver Russell syndrome in a preterm girl with 8q12.1 deletion encompassing PLAG1. Clin Dysmorphol. 2021 Oct 1;30(4):194-196. doi: 10.1097/MCD.0000000000000375. PMID: 34480472.

Published
2021

3. Rare missense variant inMSH4associated with primary gonadal failure in both 46, XX and 46, XY individuals

Author

Mehdi Totonchi, Mehri Mashayekhi, Ken McElreavey, Navid Almadani, Anu Bashambou, Arvand Akbari, Kimiya Padidar, Najmeh Salehi, Mohammad Ali Sadighi Gilani, Royan Institute for Reproductive Biomedicine [Tehran, Iran], Academic Center for Education, Culture and Research (ACECR), University of Tehran, Génétique du Développement humain - Human developmental genetics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This study was financially supported by Royan Institute, Tehran, Iran, and Institut Pasteur, Paris, France, We are sincerely thankful to the personnel of Royan Department of Genetics and the Human Developmental Genetics Unit of Institut Pasteur, Paris, France, and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Male, Proband, 46, XX Disorders of Sex Development, [SDV]Life Sciences [q-bio], Cell Cycle Proteins, Iran, MESH: Mice, Knockout, Mice, non-obstructive azoospermia, Missense mutation, MESH: Animals, Exome sequencing, Mice, Knockout, Sanger sequencing, Genetics, 0303 health sciences, MESH: Paris, 030305 genetics & heredity, Rehabilitation, Obstetrics and Gynecology, oligozoospermia, DNA-Binding Proteins, symbols, Female, primary gonadal failure, France, Paris, dbSNP, familial exome sequencing, Biology, 03 medical and health sciences, symbols.namesake, MESH: Cell Cycle Proteins, Animals, Humans, MESH: Mice, Gene, Retrospective Studies, 030304 developmental biology, Disorder of Sex Development, 46,XY, MESH: Humans, MESH: Retrospective Studies, MSH4, MESH: Male, primary ovarian insufficiency, MESH: France, MSH5, Reproductive Medicine, MESH: Iran, MESH: Female, MESH: DNA-Binding Proteins
Abstract

STUDY QUESTIONCan whole-exome sequencing (WES) reveal a shared pathogenic variant responsible for primary gonadal failure in both male and female patients from a consanguineous family?SUMMARY ANSWERPatients with primary ovarian insufficiency (POI) and non-obstructive azoospermia (NOA) were homozygous for the rare missense variant p. S754L located in the highly conserved MSH4 MutS signature motif of the ATPase domain. An oligozoospermic patient was heterozygous for the variant.WHAT IS KNOWN ALREADYMSH4 is a meiosis-specific protein expressed at a certain level in the testes and ovaries. Along with its heterodimer partner MSH5, it is responsible for double-strand Holliday junction recognition and stabilization, to ensure accurate chromosome segregation during meiosis. Knockout male and female mice for Msh4 and Msh5 are reportedly infertile due to meiotic arrest. In humans, MSH4 is associated with male and female gonadal failure, with distinct variations in the MutS domain V.STUDY DESIGN, SIZE, DURATIONThis was a retrospective genetics study of a consanguineous family with multiple cases of gonadal failure in both genders. The subject family was recruited in Iran, in 2018.PARTICIPANTS/MATERIALS, SETTING, METHODSThe proband who is affected by POI, an NOA brother, a fertile sister and their parents were subjected to WES. The discovered variant was validated in these individuals, and the rest of the family was also genotyped by Sanger sequencing. The variant was not detected in 800 healthy Iranian individuals from the Iranome database nor in 30 sporadic NOA and 30 sporadic POI patients. Suggested effect in aberrant splicing was studied by RT-PCR. Moreover, protein homology modeling was used to further investigate the amino acid substitution in silico.MAIN RESULTS AND THE ROLE OF CHANCEThe discovered variant is very rare and has never been reported in the homozygous state. It occurs in the ATPase domain at Serine 754, the first residue within the highly conserved MutS signature motif, substituting it with a Leucine. All variant effect prediction tools indicated this variant as deleterious. Since the substitution occurs immediately before the Walker B motif at position 755, further investigations based on protein homology were conducted. Considering the modeling results, the nature of the substituted amino acid residue and the distances between p. S754L variation and the residues of the Walker B motif suggested the possibility of conformational changes affecting the ATPase activity of the protein.LARGE SCALE DATAWe have submitted dbSNP entry rs377712900 to ClinVar under SCV001169709, SCV001169708 and SCV001142647 for oligozoospermia, NOA and POI, respectively.LIMITATIONS, REASONS FOR CAUTIONStudies in model organisms can shed more light on the role of this variant as our results were obtained by variant effect prediction tools and protein homology modeling.WIDER IMPLICATIONS OF THE FINDINGSIdentification of variants in meiotic genes should improve genetic counseling for both male and female infertility. Also, as two of our NOA patients underwent testicular sperm extraction (TESE) with no success, ruling out the existence of pathogenic variants in meiotic genes in such patients prior to TESE could prove useful.STUDY FUNDING/COMPETING INTEREST(S)This study was financially supported by Royan Institute in Tehran, Iran, and Institut Pasteur in Paris, France. The authors declare no competing interests.TRIAL REGISTRATION NUMBERN/A

Published
2021

4. Autosomal Recessive Cerebellar Ataxias With Elevated Alpha‐Fetoprotein: Uncommon Diseases, Common Biomarker

Author

Mathilde Renaud, Christine Tranchant, Michel Koenig, Mathieu Anheim, Service de Génétique Clinique [CHRU Nancy], Centre Hospitalier Régional Universitaire de Nancy (CHRU Nancy), Nutrition-Génétique et Exposition aux Risques Environnementaux (NGERE), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lorraine (UL), Service de Neurologie [Strasbourg], CHU Strasbourg-Hopital Civil, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg (UNISTRA), Laboratoire de génétique des maladies rares. Pathologie moleculaire, etudes fonctionnelles et banque de données génétiques (LGMR), Université Montpellier 1 (UM1)-IFR3, Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), and Herrada, Anthony

Subjects
MESH: Multifunctional Enzymes, Cerebellar Ataxia, MESH: DNA Helicases, MESH: Ataxia Telangiectasia, Ataxia Telangiectasia, alpha-fetoprotein, 03 medical and health sciences, 0302 clinical medicine, Cogan Syndrome, Humans, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Cogan Syndrome, 030304 developmental biology, 0303 health sciences, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, MESH: Humans, MESH: RNA Helicases, oculomotor apraxia, DNA Helicases, Nuclear Proteins, Multifunctional Enzymes, MESH: Phosphotransferases (Alcohol Group Acceptor), digestive system diseases, MESH: Cerebellar Ataxia, 3. Good health, DNA-Binding Proteins, Phosphotransferases (Alcohol Group Acceptor), DNA Repair Enzymes, MESH: DNA Repair Enzymes, recessive ataxia, Neurology, DNA/RNA repair, MESH: Biomarkers, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], alpha-Fetoproteins, MESH: alpha-Fetoproteins, Neurology (clinical), MESH: Nuclear Proteins, Biomarkers, RNA Helicases, MESH: DNA-Binding Proteins, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, 030217 neurology & neurosurgery
Abstract

International audience; alpha-Fetoprotein (AFP) is a biomarker of several autosomal recessive cerebellar ataxias (ARCAs), especially ataxia telangiectasia (AT) and ataxia with oculomotor apraxia (AOA) type 2 (AOA2). More recently, slightly elevated AFP has been reported in AOA1 and AOA4. Interestingly, AOA1, AOA2, AOA4, and AT are overlapping ARCAs characterized by oculomotor apraxia, with oculocephalic dissociation, choreo-dystonia, and/or axonal sensorimotor neuropathy, in addition to cerebellar ataxia with cerebellar atrophy. The genetic backgrounds in these disorders play central roles in nuclear maintenance through DNA repair [ATM (AT), APTX (AOA1), or PNKP (AOA4)] or RNA termination [SETX (AOA2)]. Partially discriminating thresholds of AFP have been proposed as a way to distinguish between ARCAs with elevated AFP. In these entities, elevated AFP may be an epiphenomenon as a result of liver transcriptional dysregulation. AFP is a simple and reliable biomarker for the diagnosis of ARCA in performance and interpretation of next-generation sequencing. Here, we evaluated clinical, laboratory, imaging, and molecular data of the group of ARCAs that share elevated AFP serum levels that have been described in the past two decades. © 2020 International Parkinson and Movement Disorder Society.

Published
2020

5. SMYD3 Impedes Small Cell Lung Cancer Sensitivity to Alkylation Damage through RNF113A Methylation–Phosphorylation Cross-talk

Author

Valentina Lukinović, Simone Hausmann, Gael S. Roth, Clement Oyeniran, Tanveer Ahmad, Ning Tsao, Joshua R. Brickner, Alexandre G. Casanova, Florent Chuffart, Ana Morales Benitez, Jessica Vayr, Rebecca Rodell, Marianne Tardif, Pascal W.T.C. Jansen, Yohann Couté, Michiel Vermeulen, Pierre Hainaut, Pawel K. Mazur, Nima Mosammaparast, Nicolas Reynoird, Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ANR-16-CE11-0018,S3S,Signalisation physiologique et pathologique de la lysine méthyltransférase SMYD3(2016), and Reynoird, Nicolas

Subjects
MESH: Cell Nucleus, MESH: RNA Processing, Post-Transcriptional, Lung Neoplasms, MESH: DNA Helicases, MESH: AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase, [SDV.CAN]Life Sciences [q-bio]/Cancer, Methylation, [SDV.MHEP.PSR]Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, MESH: Methylation, MESH: DNA Methylation, [SDV.CAN] Life Sciences [q-bio]/Cancer, Cell Line, Tumor, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, methylation signaling, MESH: Neoplasms, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Phosphorylation, Molecular Biology, alkylation, E3 ligase, SMYD3, MESH: Humans, MESH: R-Loop Structures, MESH: Transcription, Genetic, SCLC, Histone-Lysine N-Methyltransferase, ASCC, MESH: RNA, Neoplasm, Small Cell Lung Carcinoma, DNA-Binding Proteins, Oncology, RNF113A, MESH: HEK293 Cells, MESH: HeLa Cells, RNA methylation, [SDV.MHEP.PSR] Life Sciences [q-bio]/Human health and pathology/Pulmonology and respiratory tract, MESH: Ubiquitination, transcription, Protein Processing, Post-Translational, MESH: Nuclear Proteins, genome stability, MESH: Spliceosomes, MESH: DNA-Binding Proteins
Abstract

Small cell lung cancer (SCLC) is the most fatal form of lung cancer, with dismal survival, limited therapeutic options, and rapid development of chemoresistance. We identified the lysine methyltransferase SMYD3 as a major regulator of SCLC sensitivity to alkylation-based chemotherapy. RNF113A methylation by SMYD3 impairs its interaction with the phosphatase PP4, controlling its phosphorylation levels. This cross-talk between posttranslational modifications acts as a key switch in promoting and maintaining RNF113A E3 ligase activity, essential for its role in alkylation damage response. In turn, SMYD3 inhibition restores SCLC vulnerability to alkylating chemotherapy. Our study sheds light on a novel role of SMYD3 in cancer, uncovering this enzyme as a mediator of alkylation damage sensitivity and providing a rationale for small-molecule SMYD3 inhibition to improve responses to established chemotherapy. Significance: SCLC rapidly becomes resistant to conventional chemotherapy, leaving patients with no alternative treatment options. Our data demonstrate that SMYD3 upregulation and RNF113A methylation in SCLC are key mechanisms that control the alkylation damage response. Notably, SMYD3 inhibition sensitizes cells to alkylating agents and promotes sustained SCLC response to chemotherapy. This article is highlighted in the In This Issue feature, p. 2007

Published
2022

6. Aberrant RNA methylation triggers recruitment of an alkylation repair complex

Author

Alexandre G. Casanova, Li-Sheng Zhang, Ning Tsao, Brittany A. Townley, Nima Mosammaparast, Joshua R. Brickner, Chuan He, John A. Tainer, Rebecca Rodell, Tanveer Ahmad, Matthew D. Wood, Hua Sun, Jennifer M. Soll, Nicolas Reynoird, Clement Oyeniran, Alessandro Vindigni, Adit Ganguly, Albino Bacolla, Valentina Lukinović, Washington University School of Medicine [Saint Louis, MO], Washington University School of Medicine in St. Louis, Washington University in Saint Louis (WUSTL), Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), The University of Texas M.D. Anderson Cancer Center [Houston], University of Chicago, and Reynoird, Nicolas

Subjects
Transcription, Genetic, [SDV]Life Sciences [q-bio], MESH: DNA Helicases, MESH: AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase, Alkylation, MESH: DNA Methylation, Transcription (biology), Neoplasms, Sense (molecular biology), MESH: Neoplasms, RNA, Neoplasm, RNA Processing, Post-Transcriptional, E3 ligase, biology, MESH: R-Loop Structures, Nuclear Proteins, Ubiquitin ligase, Cell biology, RNF113A, [SDV] Life Sciences [q-bio], DNA-Binding Proteins, DNA Alkylation, MESH: HEK293 Cells, RNA methylation, AlkB Homolog 3, Alpha-Ketoglutarate-Dependent Dioxygenase, transcription, MESH: Spliceosomes, MESH: Cell Nucleus, MESH: RNA Processing, Post-Transcriptional, Methylation, Article, MESH: Methylation, Humans, Molecular Biology, alkylation, Cell Nucleus, MESH: Humans, MESH: Transcription, Genetic, DNA Helicases, Ubiquitination, RNA, Cell Biology, ASCC, DNA Methylation, MESH: RNA, Neoplasm, HEK293 Cells, MESH: HeLa Cells, biology.protein, Spliceosomes, MESH: Ubiquitination, R-Loop Structures, MESH: Nuclear Proteins, genome stability, MESH: DNA-Binding Proteins, HeLa Cells
Abstract

International audience; Summary Central to genotoxic responses is their ability to sense highly specific signals to activate the appropriate repair response. We previously reported that the activation of the ASCC-ALKBH3 repair pathway is exquisitely specific to alkylation damage in human cells. Yet the mechanistic basis for the selectivity of this pathway was not immediately obvious. Here, we demonstrate that RNA but not DNA alkylation is the initiating signal for this process. Aberrantly methylated RNA is sufficient to recruit ASCC, while an RNA dealkylase suppresses ASCC recruitment during chemical alkylation. In turn, recruitment of ASCC during alkylation damage, which is mediated by the E3 ubiquitin ligase RNF113A, suppresses transcription and R-loop formation. We further show that alkylated pre-mRNA is sufficient to activate RNF113A E3 ligase in vitro in a manner dependent on its RNA binding Zn-finger domain. Together, our work identifies an unexpected role for RNA damage in eliciting a specific response to genotoxins.

Published
2021

7. MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair

Author

de Krijger, Inge, Föhr, Bastian, Pérez, Santiago Hernández, Vincendeau, Estelle, Serrat, Judit, Thouin, Alexander Marc, Susvirkar, Vivek, Lescale, Chloé, Paniagua, Inés, Hoekman, Liesbeth, Kaur, Simranjeet, Altelaar, Maarten, Deriano, Ludovic, Faesen, Alex C, Jacobs, Jacqueline J L, Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Netherlands Cancer Institute (NKI), Antoni van Leeuwenhoek Hospital, Max Planck Institute for Biophysical Chemistry (MPI-BPC), Max-Planck-Gesellschaft, Université Paris Diderot, Sorbonne Paris Cité, Paris, France, Université Paris Diderot - Paris 7 (UPD7), Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Physiopathologie du système immunitaire (Inserm U1223), Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University [Utrecht], This work was supported by project grant 10999/2017-1 from the Dutch Cancer Society (KWF) to J.J.L.J., by the Institut Pasteur to L.D., by the Institut National Du Cancer (INCA_13852) to L.D., by the Ligue Nationale Contre le Cancer (Equipe labellisée 2019) to L.D. The Proteomics Facility of the Netherlands Cancer Institute was supported by the X-omics Initiative, partially funded by the Dutch Research Council (NWO)(project 184.034.019). E.V. received funding from Université de Paris, Sorbonne Paris Cité. A.C.F. is grateful for generous core funding by the Max Planck Society., Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Université Sorbonne Paris Cité (USPC), Afd Biomol.Mass Spect. and Proteomics, Biomolecular Mass Spectrometry and Proteomics, Institut Pasteur [Paris], Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris], and Lescale, Chloé

Subjects
DNA Repair, Chemistry(all), ATPase, MESH: DNA Breaks, Double-Stranded, Gene Expression, General Physics and Astronomy, Cell Cycle Proteins, [SDV.GEN] Life Sciences [q-bio]/Genetics, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Biochemistry, Piperazines, MESH: Recombinant Proteins, Mice, chemistry.chemical_compound, 0302 clinical medicine, DNA Breaks, Double-Stranded, MESH: Animals, 0303 health sciences, Multidisciplinary, biology, Chemistry, MESH: Protein Multimerization, DNA damage and repair, MESH: DNA, Recombinant Proteins, 3. Good health, Cell biology, DNA-Binding Proteins, MESH: ATPases Associated with Diverse Cellular Activities, MESH: HEK293 Cells, Mad2 Proteins, MESH: Phthalazines, MESH: Mad2 Proteins, Protein Binding, MESH: Cell Line, Tumor, MESH: Gene Expression, DNA repair, Science, Physics and Astronomy(all), Article, Chromosomes, General Biochemistry, Genetics and Molecular Biology, Cell Line, 03 medical and health sciences, MESH: Cell Cycle Proteins, Cell Line, Tumor, Animals, Humans, MESH: Protein Binding, Protein Interaction Domains and Motifs, MESH: Mice, 030304 developmental biology, MESH: Protein Interaction Domains and Motifs, [SDV.GEN]Life Sciences [q-bio]/Genetics, Binding Sites, MESH: Humans, TRIP13, Biochemistry, Genetics and Molecular Biology(all), [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, DNA, General Chemistry, DNA Repair Pathway, Fibroblasts, MESH: Cell Line, HEK293 Cells, MESH: Binding Sites, MESH: Cisplatin, MESH: Fibroblasts, MESH: HeLa Cells, biology.protein, ATPases Associated with Diverse Cellular Activities, Phthalazines, Cisplatin, Protein Multimerization, 030217 neurology & neurosurgery, Function (biology), MESH: DNA-Binding Proteins, HeLa Cells, Genetics and Molecular Biology(all)
Abstract

MAD2L2 (REV7) plays an important role in DNA double-strand break repair. As a member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization-surface, HORMA-domain protein MAD2L2 has the extraordinary ability to wrap its C-terminus around SHLD3, likely creating a very stable complex. We show that appropriate function of MAD2L2 within shieldin requires its dimerization, mediated by SHLD2 and accelerating MAD2L2-SHLD3 interaction. Dimerization-defective MAD2L2 impairs shieldin assembly and fails to promote NHEJ. Moreover, MAD2L2 dimerization, along with the presence of SHLD3, allows shieldin to interact with the TRIP13 ATPase, known to drive topological switches in HORMA-domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity., MAD2L2 — a member of the shieldin complex — is known to play important roles in DNA repair. Here the authors demonstrate how MAD2L2 dimerization mediated through SHLD2 participates in shieldin assembly and function.

Published
2021

8. Single-Stranded DNA-Binding Proteins in the Archaea

Author

Simonetta Gribaldo, Najwa Taib, Stuart A. MacNeill, Biologie Évolutive de la Cellule Microbienne - Evolutionary Biology of the Microbial Cell, Université Paris Cité (UPCité)-Microbiologie Intégrative et Moléculaire (UMR6047), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, Institut Pasteur [Paris] (IP)-Université Paris Cité (UPCité), School of Biology [University of St Andrews], and University of St Andrews [Scotland]

Subjects
DNA repair, Protein subunit, Replication protein A, MESH: DNA Replication, Computational biology, DNA replication, [SDV.BID.SPT]Life Sciences [q-bio]/Biodiversity/Systematics, Phylogenetics and taxonomy, 03 medical and health sciences, Phylogenetics, Three-domain system, ssDNA, MESH: Protein Binding, MESH: Species Specificity, MESH: Phylogeny, SSB, 030304 developmental biology, MESH: DNA Repair, 0303 health sciences, biology, MESH: DNA, Archaeal, 030302 biochemistry & molecular biology, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: Archaeal Proteins, biology.organism_classification, Archaea, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, enzymes and coenzymes (carbohydrates), MESH: DNA, Single-Stranded, OB fold, MESH: Archaea, MESH: Protein Domains, Single-stranded DNA, Bacteria, RPA, MESH: DNA-Binding Proteins, MESH: Models, Molecular
Abstract

International audience; Single-stranded (ss) DNA-binding proteins are found in all three domains of life where they play vital roles in nearly all aspects of DNA metabolism by binding to and stabilizing exposed ssDNA and acting as platforms onto which DNA-processing activities can assemble. The ssDNA-binding factors SSB and RPA are extremely well conserved across bacteria and eukaryotes, respectively, and comprise one or more OB-fold ssDNA-binding domains. In the third domain of life, the archaea, multiple types of ssDNA-binding protein are found with a variety of domain architectures and subunit compositions, with OB-fold ssDNA-binding domains being a characteristic of most, but not all. This chapter summarizes current knowledge of the distribution, structure, and biological function of the archaeal ssDNA-binding factors, highlighting key features shared between clades and those that distinguish the proteins of different clades from one another. The likely cellular functions of the proteins are discussed and gaps in current knowledge identified.

Published
2021

9. Interplay between Histone and DNA Methylation Seen through Comparative Methylomes in Rare Mendelian Disorders

Author

Guillaume Velasco, Damien Ulveling, Sophie Rondeau, Pauline Marzin, Motoko Unoki, Valérie Cormier-Daire, Claire Francastel, Gestionnaire, Hal Sorbonne Université, Une maladie rare pour décoder le lien fonctionnel entre méthylation de l'ADN et maintien de l'intégrité du centromère - - MEliCENDre2019 - ANR-19-CE12-0022 - AAPG2019 - VALID, Centre épigénétique et destin cellulaire (EDC (UMR_7216)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Kyushu University, This research was funded by the Agence Nationale pour la Recherche (grant ANR-19-CE12-0022) and the Fondation Maladies Rares (GenOmics of Rare Diseases, call 2017)., ANR-19-CE12-0022,MEliCENDre,Une maladie rare pour décoder le lien fonctionnel entre méthylation de l'ADN et maintien de l'intégrité du centromère(2019), Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), and Kyushu University [Fukuoka]

Subjects
MESH: Neoplasm Proteins, MESH: Rare Diseases, MESH: DNA (Cytosine-5-)-Methyltransferases, MESH: Genetic Diseases, Inborn, MESH: Mutation, [SDV]Life Sciences [q-bio], epigenetic crosstalks, Article, DNA Methyltransferase 3A, lcsh:Chemistry, Histones, MESH: DNA Methylation, Humans, DNA (Cytosine-5-)-Methyltransferases, histone methylation, lcsh:QH301-705.5, MESH: Histones, MESH: Humans, Genetic Diseases, Inborn, MESH: Histone-Lysine N-Methyltransferase, rare diseases, biomarkers, Histone-Lysine N-Methyltransferase, DNA Methylation, Neoplasm Proteins, DNA-Binding Proteins, [SDV] Life Sciences [q-bio], lcsh:Biology (General), lcsh:QD1-999, Mutation, DNA methylation profiling, MESH: DNA-Binding Proteins
Abstract

International audience; DNA methylation (DNAme) profiling is used to establish specific biomarkers to improve the diagnosis of patients with inherited neurodevelopmental disorders and to guide mutation screening. In the specific case of mendelian disorders of the epigenetic machinery, it also provides the basis to infer mechanistic aspects with regard to DNAme determinants and interplay between histone and DNAme that apply to humans. Here, we present comparative methylomes from patients with mutations in the de novo DNA methyltransferases DNMT3A and DNMT3B, in their catalytic domain or their N-terminal parts involved in reading histone methylation, or in histone H3 lysine (K) methylases NSD1 or SETD2 (H3 K36) or KMT2D/MLL2 (H3 K4). We provide disease-specific DNAme signatures and document the distinct consequences of mutations in enzymes with very similar or intertwined functions, including at repeated sequences and imprinted loci. We found that KMT2D and SETD2 germline mutations have little impact on DNAme profiles. In contrast, the overlapping DNAme alterations downstream of NSD1 or DNMT3 mutations underlines functional links, more specifically between NSD1 and DNMT3B at heterochromatin regions or DNMT3A at regulatory elements. Together, these data indicate certain discrepancy with the mechanisms described in animal models or the existence of redundant or complementary functions unforeseen in humans. View Full-Text

Published
2021
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10. A structural inventory of native ribosomal ABCE1‐43S pre‐initiation complexes

Author

Thomas Becker, Otto Berninghausen, Timur Mackens-Kiani, Roland Beckmann, Abdelkader Namane, Franz Herzog, Mia Potocnjak, Estelle Dacheux, Jingdong Cheng, Hanna Kratzat, Michael Ameismeier, Micheline Fromont-Racine, Ludwig-Maximilians-Universität München (LMU), Génétique des Interactions macromoléculaires, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by German Research Council (BE1814/15-1 and TRR174), the Center for Integrated Protein Science Munich (CiPS-M), the ANR-17-CE11-0049-01 and the ANR-17-CE12-0024-02 grants, the Pasteur Institute and the Centre National de la Recherche Scientifique. H.K., M.A., and M.P. are supported by a DFG fellowship through the Graduate School of Quantitative Bioscience Munich (QBM). Open Access funding enabled and organized by ProjektDEAL., ANR-17-CE11-0049,SKA,Caracterisation structurale et fonctionnelle d'un sous-complexe SKI chez Saccharomyces cerevisiae(2017), ANR-17-CE12-0024,Rescue_ribosome,Voies de sauvetage pour les ribosomes non recyclés(2017), Génétique des Interactions macromoléculaires / Genetics of Macromolecular Interactions, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Eukaryotic Initiation Factor-2, ABCE1, [SDV]Life Sciences [q-bio], Eukaryotic Initiation Factor-2, 0302 clinical medicine, MESH: Saccharomyces cerevisiae Proteins, RNA, Transfer, Structural Biology, Ternary complex, cryo‐EM, 0303 health sciences, eIF2, General Neuroscience, Translation (biology), Articles, MESH: Saccharomyces cerevisiae, 3. Good health, Cell biology, DNA-Binding Proteins, MESH: Protein Biosynthesis, MESH: HEK293 Cells, Transfer RNA, MESH: ATP-Binding Cassette Transporters, eIF3, Protein Binding, ribosome recycling, Saccharomyces cerevisiae Proteins, Context (language use), Saccharomyces cerevisiae, Biology, MESH: Ribosome Subunits, Small, Eukaryotic, translation initiation, General Biochemistry, Genetics and Molecular Biology, Article, Cell Line, 03 medical and health sciences, Eukaryotic translation, Humans, MESH: Protein Binding, Eukaryotic Small Ribosomal Subunit, Translation & Protein Quality, RNA, Messenger, Molecular Biology, 030304 developmental biology, MESH: RNA, Messenger, Ribosome Subunits, Small, Eukaryotic, MESH: Humans, General Immunology and Microbiology, MESH: RNA, Transfer, MESH: Cell Line, HEK293 Cells, Protein Biosynthesis, biology.protein, cryo-EM, ATP-Binding Cassette Transporters, 030217 neurology & neurosurgery, MESH: DNA-Binding Proteins
Abstract

In eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub‐complexes. These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP‐binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre‐initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1‐containing pre‐initiation complexes by cryo‐EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide‐binding domains, while interacting with the N‐terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C‐terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near‐complete molecular picture of the architecture and sophisticated interaction network of the 43S‐bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA., Cryo‐EM structures of native human and yeast translation initiation complexes reveal a novel hybrid conformation of the recycling factor ATPase ABCE1, which is stabilized by a dimer of translation initiation factor eIF3j.

Published
2021

11. Repair of G1 induced DNA double-strand breaks in S-G2/M by alternative NHEJ

Author

Loelia Babin, Caroline Demangel, Hélène Lenden-Hasse, Ludovic Deriano, Ludivine Baron, Erika Brunet, José Yélamos, Marie Bedora-Faure, Wei Yu, Chloé Lescale, Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris], Genome dynamics in the immune system (Equipe Inserm U1163), Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Immunobiologie de l'Infection - Immunobiology of Infection, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), IMIM-Hospital del Mar, Generalitat de Catalunya, This project is funded by the Institut Pasteur (L.D. and C.D. labs), the Institut National du Cancer (INCa Grant # PLBIO16-181 to L.D. and E.B. labs), the Ligue Nationale Contre le Cancer (Équipe Labellisée 2019 L.D. lab and Équipe Labellisée 2017 and 2020 E.B. lab) the Cancéropôle IdF-INCa (Emergence 2016 grant to L.D. and C.D.), the Spanish Ministerio de Economía, Industria y Competitividad (Grant SAF2017-83565-R to J.Y.) as well as by the Fundación Científica de la Asociación Española Contra el Cáncer (AECC) (Grant PROYEI6018YÉLA to J.Y.)., We thank the Institut Pasteur genomics and cytometry platforms for help with sequencing and cell sorting, Frederick Alt and members of his lab for tremendous help with LAM-HTGTS experiments, Barry Sleckman for providing the RAG2-Thy1.1 complementation vector, Joy Bianchi for providing the Rag2−/− p53−/− v-Abl pro-B cell lines, Carine Giovannangeli and Anne de Cian for providing the Cas9 protein and Thomas Mercher for helpful discussions and suggestions., Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Demangel, Caroline, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur [Paris]

Subjects
0301 basic medicine, Genome instability, DNA End-Joining Repair, MESH: DNA Breaks, Double-Stranded, Poly (ADP-Ribose) Polymerase-1, General Physics and Astronomy, MESH: Cell Cycle, MESH: Poly (ADP-Ribose) Polymerase-1, Genome, Càncer--Tractament, chemistry.chemical_compound, Mice, 0302 clinical medicine, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, DNA Breaks, Double-Stranded, MESH: Animals, lcsh:Science, Double strand, Multidisciplinary, Cell Cycle, DNA repair protein XRCC4, Cell cycle, 3. Good health, Cell biology, DNA-Binding Proteins, 030220 oncology & carcinogenesis, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Cell division, DNA repair, ADN--Dany, DNA recombination, Science, Double-strand DNA breaks, MESH: G1 Phase, General Biochemistry, Genetics and Molecular Biology, Article, 03 medical and health sciences, Targeted therapies, Animals, MESH: Mice, B cells, fungi, G1 Phase, General Chemistry, MESH: DNA End-Joining Repair, enzymes and coenzymes (carbohydrates), 030104 developmental biology, chemistry, lcsh:Q, Homologous recombination, DNA, MESH: DNA-Binding Proteins
Abstract

The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies., Depending on the cell cycle stage, cells can repair their genome via different pathways. Here the authors reveal mechanistic insights into repair of double strand breaks induced during G1 in an error-prone manner by Pol θ-dependent and PARP1-independent alt NHEJ during the SG2/M phases of the cell cycle

Published
2020

12. Mycobacterium tuberculosis FasR senses long fatty acyl-CoA through a tunnel and a hydrophobic transmission spine

Author

Marisa M. Fernández, Felipe Trajtenberg, Hugo Gramajo, Lautaro Diacovich, Nicole Larrieux, Gabriela Gago, Julia Lara, Emilio L. Malchiodi, Alejandro Buschiazzo, Universidad Nacional de Rosario [Santa Fe], Plataforma de Biología Estructural y Metabolómica [Rosario] (PLABEM), Molecular and structural microbiology / Microbiología Molecular y Estructural [Montevideo], Institut Pasteur de Montevideo, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Universidad de Buenos Aires [Buenos Aires] (UBA), Integrative Microbiology of Zoonotic Agents [Paris and Montevideo] (IMiZA), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur [Paris], J.L. traineeships at IPasteur-Montevideo were funded by CeBEM. Support to A.B. from Institut Pasteur (grant 761-International_Joint_Research_Unit-IMiZA-2016), to G.G. from ANPCyT (grant PICT 2015-0796) and to H.G. from ANPCyT (grants PICT 2012-0168 and 2022) and NIH (grant 1R01AI095183-01) are acknowledged., We thank Stanislas Leibler, Michael Mitchell and Pablo Sartori for sharing initial strain analysis scripts, Matias Machado for assistance in molecular dynamics, Frank Lehmann for initial cloning efforts, Sebastian Klinke (Fundación Leloir) and the staff at Proxima 1 beamline (Soleil synchrotron) and at I04-1 beamline (Diamond synchrotron) for assistance with data collection. We acknowledge computational and storage services (TARS cluster) provided by the Institut Pasteur IT Dept (Paris). We thank the CCP4/CeBEM Macromolecular Crystallography School (USP@São Carlos, 2018), especially Isabel Usón and Paul Emsley for helping us, respectively, with ShelxE and Coot, in dealing with low-resolution density modification and model building., and Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut Pasteur [Paris] (IP)

Subjects
Models, Molecular, 0301 basic medicine, MESH: Mycobacterium tuberculosis, Protein Conformation, General Physics and Astronomy, Crystallography, X-Ray, Ligands, chemistry.chemical_compound, Protein structure, MESH: Protein Conformation, Cell Wall, MESH: Ligands, lcsh:Science, MESH: Allosteric Site, MESH: Bacterial Proteins, Multidisciplinary, biology, Effector, Fatty Acids, MESH: Transcription Factors, 3. Good health, Cell biology, MESH: Fatty Acids, DNA-Binding Proteins, Structural biology, Transcription, Allosteric Site, MESH: Models, Molecular, DNA, Bacterial, Science, Allosteric regulation, Protein dimer, Bacterial physiology, Article, General Biochemistry, Genetics and Molecular Biology, Mycobacterium tuberculosis, 03 medical and health sciences, Acyl-CoA, MESH: Cell Wall, Bacterial Proteins, Lipid biosynthesis, MESH: Acyl Coenzyme A, [CHIM.CRIS]Chemical Sciences/Cristallography, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030102 biochemistry & molecular biology, General Chemistry, biology.organism_classification, MESH: Crystallography, X-Ray, MESH: DNA, Bacterial, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 030104 developmental biology, chemistry, lcsh:Q, Acyl Coenzyme A, DNA, MESH: DNA-Binding Proteins, Transcription Factors
Abstract

Mycobacterium tuberculosis is a pathogen with a unique cell envelope including very long fatty acids, implicated in bacterial resistance and host immune modulation. FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis. Here we disclose crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms. A long tunnel traverses the entire effector-binding domain, enabling long fatty acyl effectors to bind. Only when the tunnel is entirely occupied, the protein dimer adopts a rigid configuration with its DNA-binding domains in an open state, leading to DNA dissociation. The protein-folding hydrophobic core connects the two domains, and is completed into a continuous spine when the effector binds. Such a transmission spine is conserved in a large number of TetR-like regulators, offering insight into effector-triggered allosteric functional control., FasR is a TetR-like transcriptional activator that plays a central role in sensing mycobacterial long-chain fatty acids and regulating lipid biosynthesis in Mycobacterium tuberculosis. Here authors present crystal structures of M. tuberculosis FasR in complex with acyl effector ligands and with DNA, uncovering its molecular sensory and switching mechanisms.

Published
2020

13. Resection and repair of a Cas9 double-strand break at CTG trinucleotide repeats induces local and extensive chromosomal deletions

Author

Wilhelm Vaysse-Zinkhöfer, David Viterbo, Stéphane Descorps-Declère, Lucie Poggi, Guy-Franck Richard, Valentine Mosbach, Génétique des génomes - Genetics of Genomes (UMR 3525), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), Collège Doctoral, Sorbonne Université (SU), and V. M. was supported by Fondation Guy Nicolas and Fondation Hardy. W. V.-Z. is the recipient of a PhD fellowship from la Ligue Nationale Contre le Cancer. This work was generously supported by the Institut Pasteur and by the Centre National de la Recherche Scientifique (CNRS).

Subjects
Cancer Research, MESH: CRISPR-Cas Systems, DNA End-Joining Repair, DNA Repair, [SDV]Life Sciences [q-bio], RAD52, MESH: DNA Breaks, Double-Stranded, Yeast and Fungal Models, MESH: CRISPR-Associated Protein 9, QH426-470, Biochemistry, Genome, Electrophoretic Blotting, DNA Ligase ATP, Guide RNA, MESH: Saccharomyces cerevisiae Proteins, Trinucleotide Repeats, CRISPR-Associated Protein 9, MESH: Endonucleases, MESH: Gene Conversion, MESH: Trinucleotide Repeat Expansion, Myotonic Dystrophy, DNA Breaks, Double-Stranded, Genetics (clinical), Gel Electrophoresis, Recombination, Genetic, Genetics, MESH: DNA Repair, Fungal genetics, Eukaryota, MESH: Saccharomyces cerevisiae, MESH: Chromosomes, Fungal, DNA-Binding Proteins, Nucleic acids, Experimental Organism Systems, Saccharomyces Cerevisiae, MESH: Recombination, Genetic, Chromosome Deletion, Chromosomes, Fungal, Research Article, MESH: Trinucleotide Repeats, Saccharomyces cerevisiae Proteins, MESH: Myotonic Dystrophy, MESH: Chromosome Deletion, Gene Conversion, Molecular Probe Techniques, Biology, Research and Analysis Methods, Myotonic dystrophy, MESH: Rad52 DNA Repair and Recombination Protein, Saccharomyces, Electrophoretic Techniques, Model Organisms, MESH: DNA Ligase ATP, medicine, Humans, Gene conversion, Molecular Biology Techniques, Molecular Biology, Ecology, Evolution, Behavior and Systematics, MESH: Genome, Human, MESH: Humans, Biology and life sciences, Genome, Human, Organisms, Fungi, DNA, MESH: DNA End-Joining Repair, Endonucleases, medicine.disease, Yeast, Rad52 DNA Repair and Recombination Protein, Genetic Loci, Rad50, Mutation, Animal Studies, RNA, Human genome, CRISPR-Cas Systems, Trinucleotide Repeat Expansion, Trinucleotide repeat expansion, Southern Blot, MESH: DNA-Binding Proteins, Cloning
Abstract

Microsatellites are short tandem repeats, ubiquitous in all eukaryotes and represent ~2% of the human genome. Among them, trinucleotide repeats are responsible for more than two dozen neurological and developmental disorders. Targeting microsatellites with dedicated DNA endonucleases could become a viable option for patients affected with dramatic neurodegenerative disorders. Here, we used the Streptococcus pyogenes Cas9 to induce a double-strand break within the expanded CTG repeat involved in myotonic dystrophy type 1, integrated in a yeast chromosome. Repair of this double-strand break generated unexpected large chromosomal deletions around the repeat tract. These deletions depended on RAD50, RAD52, DNL4 and SAE2, and both non-homologous end-joining and single-strand annealing pathways were involved. Resection and repair of the double-strand break (DSB) were totally abolished in a rad50Δ strain, whereas they were impaired in a sae2Δ mutant, only on the DSB end containing most of the repeat tract. This observation demonstrates that Sae2 plays significant different roles in resecting a DSB end containing a repeated and structured sequence as compared to a non-repeated DSB end. In addition, we also discovered that gene conversion was less efficient when the DSB could be repaired using a homologous template, suggesting that the trinucleotide repeat may interfere with gene conversion too. Altogether, these data show that SpCas9 may not be the best choice when inducing a double-strand break at or near a microsatellite, especially in mammalian genomes that contain many more dispersed repeated elements than the yeast genome., Author summary With the discovery of highly specific DNA endonucleases such as TALEN and CRISPR-Cas systems, gene editing has become an attractive approach to address genetic disorders. Myotonic dystrophy type 1 (Steinert disease) is due to a large expansion of a CTG trinucleotide repeat in the DMPK gene. At the present time, despite numerous therapeutic attempts, this dramatic neurodegenerative disorder still has no cure. In the present work, we tried to use the Cas9 endonuclease to induce a double-strand break within the expanded CTG repeat of the DMPK gene integrated in the yeast genome. Surprisingly, this break induced chromosomal deletions around the repeat tract. These deletions were local and involved non-homologous joining of the two DNA ends, or more extensive involving homologous recombination between repeated elements upstream and downstream the break. Using yeast genetics, we investigated the genetic requirements for these deletions and found that the triplet repeat tract altered the capacity of the repair machinery to faithfully repair the double-strand break. These results have implications for future gene therapy approaches in human patients.

Published
2020

14. Developing a tetO/TetR system in Neurospora crassa

Author

Eugene Gladyshev, Tinh-Suong Nguyen, Epigénomique fongique - Fungal Epigenomics, Institut Pasteur [Paris] (IP), The work was supported by grants from the Agence Nationale de la Recherche (10-LABX-0062) and Fondation pour la Recherche Médicale (AJE20180539525)., ANR-10-LABX-0062,IBEID,Integrative Biology of Emerging Infectious Diseases(2010), and Institut Pasteur [Paris]

Subjects
Photomicrography, TetR, Operator Regions, Genetic, MESH: Genes, Synthetic, FROS, MESH: Neurospora crassa, MESH: Tetracycline, Green fluorescent protein, chemistry.chemical_compound, Gene Expression Regulation, Fungal, Genes, Synthetic, Fluorescence microscope, Homologous Recombination, 0303 health sciences, biology, Cell biology, DNA-Binding Proteins, MESH: Repressor Proteins, MESH: Genome, Fungal, MESH: Fungal Proteins, Genome, Fungal, Fluorescent repressor operator system, MESH: Gene Expression Regulation, Fungal, MESH: Photomicrography, Recombinant Fusion Proteins, Genes, Fungal, Microbiology, Neurospora, Neurospora crassa, Fungal Proteins, MESH: Homologous Recombination, 03 medical and health sciences, Genetics, Homologous chromosome, MESH: Recombinant Fusion Proteins, Point Mutation, Gene, tetO, Repetitive Sequences, Nucleic Acid, 030304 developmental biology, MESH: Point Mutation, MESH: Repetitive Sequences, Nucleic Acid, 030306 microbiology, Point mutation, fungi, MESH: Saporins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Tetracycline, biology.organism_classification, Saporins, Repressor Proteins, chemistry, RIP, MESH: Operator Regions, Genetic, Repeat-induced point mutation, MESH: Genes, Fungal, MESH: DNA-Binding Proteins
Abstract

International audience; The development of a tetO/TetR system in the fungus Neurospora crassa is described. The system includes (i) a synthetic gene encoding a TetR variant fused to GFP, and (ii) a standard tetO array integrated homologously, as a proof of principle, near the his-3 gene. The localization of TetR-GFP at the tetO array (observed by fluorescence microscopy) can be disrupted by the application of tetracycline. The full-length array is stable during vegetative growth, but it triggers strong repeat-induced point mutation (RIP) by the RID-dependent as well as the DIM-2-dependent pathways during the sexual phase. Thus, both RIP pathways must be inactivated to allow the faithful inheritance of the unmodified construct. In summary, this study introduces a new molecular tool into Neurospora research, and suggests that the standard tetO array can self-engage in recombination-independent homologous pairing.

Published
2020

15. Design, synthesis, and biological evaluation of dual targeting inhibitors of histone deacetylase 6/8 and bromodomain BRPF1

Author

Manfred Jung, Karin Schmidtkunz, Elizabeth R Morales, Stefan Günther, Wolfgang Sippl, Martin Hügle, Frank Erdmann, Patrik Zeyen, Ehab Ghazy, Dina Robaa, Christophe Romier, Matthias Schmidt, Daniel Herp, Martin-Luther-Universität Halle Wittenberg (MLU), Albert-Ludwigs-Universität Freiburg, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Romier, Christophe

Subjects
Bromodomain, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, MESH: Drug Design, Histone Deacetylase 6, 01 natural sciences, MESH: Structure-Activity Relationship, Drug Discovery, MESH: Molecular Dynamics Simulation, MESH: Histone Deacetylase Inhibitors, 0303 health sciences, biology, Chemistry, Acetylation, General Medicine, Hydroxamic acids, 3. Good health, Cell biology, DNA-Binding Proteins, Molecular Docking Simulation, Leukemia, Myeloid, Acute, Histone, MESH: Repressor Proteins, Epigenetics, MESH: Leukemia, Myeloid, Acute, MESH: Acetylation, MESH: Cell Line, Tumor, [SDV.SP.MED] Life Sciences [q-bio]/Pharmaceutical sciences/Medication, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, Antineoplastic Agents, [SDV.CAN]Life Sciences [q-bio]/Cancer, Molecular Dynamics Simulation, Histone Deacetylases, 03 medical and health sciences, Structure-Activity Relationship, [SDV.SP.MED]Life Sciences [q-bio]/Pharmaceutical sciences/Medication, [SDV.CAN] Life Sciences [q-bio]/Cancer, Cell Line, Tumor, MESH: Molecular Docking Simulation, Humans, Dual targeting inhibitors, 030304 developmental biology, Adaptor Proteins, Signal Transducing, MESH: Adaptor Proteins, Signal Transducing, Pharmacology, Acute myeloid leukemia, MESH: Humans, 010405 organic chemistry, Organic Chemistry, HDAC8, HDAC6, 0104 chemical sciences, Histone Deacetylase Inhibitors, Repressor Proteins, MESH: Histone Deacetylases, Docking (molecular), PHD finger, BRPF1, Drug Design, biology.protein, MESH: Antineoplastic Agents, MESH: Histone Deacetylase 6, MESH: DNA-Binding Proteins
Abstract

International audience; Histone modifying proteins, specifically histone deacetylases (HDACs) and bromodomains, have emerged as novel promising targets for anticancer therapy. In the current work, based on available crystal structures and docking studies, we designed dual inhibitors of both HDAC6/8 and the bromodomain and PHD finger containing protein 1 (BRPF1). Biochemical and biophysical tests showed that compounds 23a,b and 37 are nanomolar inhibitors of both target proteins. Detailed structure-activity relationships were deduced for the synthesized inhibitors which were supported by extensive docking and molecular dynamics studies. Cellular testing in acute myeloid leukemia (AML) cells showed only a weak effect, most probably because of the poor permeability of the inhibitors. We also aimed to analyse the target engagement and the cellular activity of the novel inhibitors by determining the protein acetylation levels in cells by western blotting (tubulin vs histone acetylation), and by assessing their effects on various cancer cell lines.

Published
2020

16. Growth charts in Kabuki syndrome 1

Author

Claire Jeandel, Caroline Michot, Odile Boute, Mario Abaji, Stanislas Lyonnet, Julie Reversat, Lucile Pinson, Sandrine Akouete, Bertrand Isidor, Marie-Ange Delrue, Valérie Cormier-Daire, Isabelle Touitou, Pierre Sarda, Bérénice Doray, Tiffany Busa, Anne Moncla, Véra Georgescu, Alice Goldenberg, Damien Sanlaville, Cyril Amouroux, Elodie Sanchez, Mélanie Fradin, Guilaine Boursier, Didier Lacombe, Mouna Barat-Houari, Annick Toutain, Brigitte Gilbert-Dussardier, David Geneviève, Fabienne Giuliano, Kim Hanh Le Quan Sang, Joelle Roume, Marianne Till, Yves Alembick, Nicole Philip, Elise Schaefer, Marjolaine Willems, Claire Duflos, Aurélia Jacquette, Eudeline Alix, Sébastien Moutton, Adeline Bonnard, Vincent Gatinois, Marie-Pierre Cordier, Clarisse Baumann, Martine Le Merrer, Marie-Line Jacquemont, Lydie Burglen, Valentin Ruault, Laurence Faivre, Carole Corsini, Sylvie Manouvrier, Svetlana Gorokhova, Anna Pelet, Delphine Héron, Sylvie Odent, Jeanne Amiel, Département de génétique médicale, maladies rares et médecine personnalisée [CHRU Montpellier], Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Département d'Information Médicale [CHRU Montpellier], Département de génétique médicale [Hôpital de la Timone - APHM], Aix Marseille Université (AMU)-Assistance Publique - Hôpitaux de Marseille (APHM)- Hôpital de la Timone [CHU - APHM] (TIMONE)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Diagnostic Génétique [CHU Strasbourg], Université de Strasbourg (UNISTRA)-CHU Strasbourg, Laboratoire de Cytogénétique Constitutionnelle [Hospices civils de Lyon], Hospices Civils de Lyon (HCL), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon, Génétique et épigénétique des maladies métaboliques, neurosensorielles et du développement (Inserm U781), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Hôpital Robert Debré Paris, Hôpital Robert Debré, Hôpital Jeanne de Flandres, Université de Lille, Droit et Santé-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Service de génétique et embryologie médicales [CHU Trousseau], CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Hôpital Pellegrin, CHU Bordeaux [Bordeaux]-Groupe hospitalier Pellegrin, Hôpital d'Enfants [CHU Dijon], Hôpital du Bocage, Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon)-Centre Hospitalier Universitaire de Dijon - Hôpital François Mitterrand (CHU Dijon), Service de génétique clinique [Rennes], Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-CHU Pontchaillou [Rennes]-hôpital Sud, Service Génétique Médicale [CHU Poitiers], Centre hospitalier universitaire de Poitiers (CHU Poitiers), Dpt génétique médicale [CHU Nice], Centre Hospitalier Universitaire de Nice (CHU Nice), Département de génétique [CHU Rouen] (Centre Normandie de Génomique et de Médecine Personnalisée), CHU Rouen, Normandie Université (NU)-Normandie Université (NU), CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Centre hospitalier universitaire de Nantes (CHU Nantes), Hôpital saint Pierre, GH Sud Réunion, Centre de référence des maladies endocriniennes rares de la croissance et du développement [CHU Pitié-Salpêtrière], Département Pédiatrie [CHRU Montpellier], Pôle Femme Mère Enfant [CHRU Montpellier], Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier)-Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Centre de recherche en neurosciences de Lyon (CRNL), Université de Lyon-Université de Lyon-Université Jean Monnet [Saint-Étienne] (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), centre hospitalier intercommunal de Poissy/Saint-Germain-en-Laye - CHIPS [Poissy], Hôpital Bretonneau, Centre Hospitalier Régional Universitaire de Tours (CHRU Tours), Département génétique méd, mal rares et médecine personnalisée [CHRU de Montpellier], Pôle Biologie-Pathologie [CHRU Montpellier], Salvy-Córdoba, Nathalie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Université de Rennes (UR)-CHU Pontchaillou [Rennes]-hôpital Sud, Centre de recherche en neurosciences de Lyon - Lyon Neuroscience Research Center (CRNL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Centre hospitalier intercommunal de Poissy/Saint-Germain-en-Laye - CHIPS [Poissy]

Subjects
Male, MESH: Neoplasm Proteins, CHILDREN, Growth, MESH: Growth Charts, Body Mass Index, MALFORMATION, Parental target size, MESH: Child, KDM6A, Growth Charts, Child, Specific curves, Genetics (clinical), 2. Zero hunger, Genetics & Heredity, Histone Demethylases, 0303 health sciences, education.field_of_study, 030305 genetics & heredity, Increase size, Circumference, MESH: Hematologic Diseases, Adult height, 3. Good health, Neoplasm Proteins, DNA-Binding Proteins, Vestibular Diseases, Child, Preschool, Cohort, MLL2, Female, MESH: Vestibular Diseases, Life Sciences & Biomedicine, MESH: Face, growth hormone deficiency, MESH: Abnormalities, Multiple, MESH: Mutation, Adolescent, growth, Population, [SDV.GEN.GH] Life Sciences [q-bio]/Genetics/Human genetics, Growth hormone deficiency, MESH: Body Mass Index, 03 medical and health sciences, [SDV.MHEP.PED] Life Sciences [q-bio]/Human health and pathology/Pediatrics, EARS, MESH: Body Height, MESH: Histone Demethylases, Genetics, medicine, Humans, Abnormalities, Multiple, education, 030304 developmental biology, MESH: Adolescent, [SDV.MHEP.PED]Life Sciences [q-bio]/Human health and pathology/Pediatrics, Science & Technology, Kabuki syndrome, MESH: Humans, business.industry, MUTATIONS, Body Weight, MESH: Child, Preschool, specific curves, MAKE-UP-SYNDROME, medicine.disease, Hematologic Diseases, Body Height, MESH: Male, MESH: Body Weight, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, parental target size, Face, Mutation, business, Body mass index, MESH: Female, MESH: DNA-Binding Proteins, Demography
Abstract

Kabuki syndrome (KS, KS1: OMIM 147920 and KS2: OMIM 300867) is caused by pathogenic variations in KMT2D or KDM6A. KS is characterized by multiple congenital anomalies and neurodevelopmental disorders. Growth restriction is frequently reported. Here we aimed to create specific growth charts for individuals with KS1, identify parameters used for size prognosis and investigate the impact of growth hormone therapy on adult height. Growth parameters and parental size were obtained for 95 KS1 individuals (41 females). Growth charts for height, weight, body mass index (BMI) and occipitofrontal circumference were generated in standard deviation values for the first time in KS1. Statural growth of KS1 individuals was compared to parental target size. According to the charts, height, weight, BMI, and occipitofrontal circumference were lower for KS1 individuals than the normative French population. For males and females, the mean growth of KS1 individuals was -2 and -1.8 SD of their parental target size, respectively. Growth hormone therapy did not increase size beyond the predicted size. This study, from the largest cohort available, proposes growth charts for widespread use in the management of KS1, especially for size prognosis and screening of other diseases responsible for growth impairment beyond a calculated specific target size. ispartof: AMERICAN JOURNAL OF MEDICAL GENETICS PART A vol:182 issue:3 pages:446-453 ispartof: location:United States status: published

Published
2019

17. Reversible induction of TDP-43 granules in cortical neurons after traumatic injury

Author

Lilla Tar, Luc Dupuis, Albert C. Ludolph, Akila Chandrasekar, William Tsao, Philip C. Wong, Birgit Linkus, Francesco Roselli, Florian olde Heuvel, Diana Wiesner, University of Ulm (UUlm), Mécanismes Centraux et Périphériques de la Neurodégénérescence, Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Johns Hopkins University School of Medicine [Baltimore], and Dieterle, Stéphane

Subjects
0301 basic medicine, Pathology, TDP-43, [SDV]Life Sciences [q-bio], Cortical injury, Mice, Superoxide Dismutase-1, 0302 clinical medicine, Brain Injuries, Traumatic, MESH: Behavior, Animal, MESH: Animals, Amyotrophic lateral sclerosis, MESH: Amyotrophic Lateral Sclerosis, MESH: Superoxide Dismutase-1, Motor Neurons, Behavior, Animal, MESH: RNA-Binding Protein FUS, Motor Cortex, Immunohistochemistry, DNA-Binding Proteins, [SDV] Life Sciences [q-bio], Traumatic injury, medicine.anatomical_structure, Neurology, MESH: Motor Neurons, Motor cortex, Genetically modified mouse, medicine.medical_specialty, MESH: Mice, Transgenic, Traumatic brain injury, SOD1, MESH: Brain Injuries, Traumatic, Mice, Transgenic, MESH: Cytoplasmic Granules, Biology, Cytoplasmic Granules, MESH: Motor Cortex, 03 medical and health sciences, Developmental Neuroscience, Autophagy, medicine, Animals, MESH: Autophagy, MESH: Mice, Amyotrophic Lateral Sclerosis, Colocalization, MESH: Immunohistochemistry, medicine.disease, nervous system diseases, Disease Models, Animal, 030104 developmental biology, RNA-Binding Protein FUS, ALS, MESH: Disease Models, Animal, MESH: DNA-Binding Proteins, 030217 neurology & neurosurgery
Abstract

International audience; Traumatic brain injury (TBI) has been proposed as a risk factor for neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). To determine whether TBI might trigger or exacerbate ALS-relevant pathology, we delivered a mild stab-wound injury to the motor cortex of three different ALS mouse models expressing mutations in SOD1, TDP-43 or FUS and scrutinized the effects on the formation of phospho-TDP-43 (pTDP-43) cytoplasmic granules. Stab-injury induced the formation of cytoplasmic TDP-43 granules in wt animals, peaking at 3dpi; a much larger response was seen in mutant TDP-43 mice, whose response peaked at 7dpi. The pTDP-43 granules did not colocalize with the stress markers TIAR-1 and FUS but colocalized with FMRP (35%) and with p62 (65%), suggesting their involvement in transport granules and their clearance by autophagy. A similar, albeit smaller effect, was seen in mutant FUS mice. In the SOD1G93A mouse model, neither increase in pTDP-43 granules nor in SOD1 aggregates were detected. In all cases, pTDP-43 granules were cleared and the number of pTDP-43-positive neurons returned to baseline by 40dpi. Neither injury-related neuronal loss nor motor performance or survival was significantly different in transgenic mice receiving injury vs sham mice. Thus, trauma can trigger ALS-related TDP-43 pathology, the extent of which is modulated by ALS-related mutations. However, the pathological findings prove reversible and do not affect disease progression and neuronal vulnerability.

Published
2018

18. Retinal Degeneration Triggers the Activation of YAP/TEAD in Reactive Müller Cells

Author

Hamon, Annaïg, Masson, Christel, Bitard, Juliette, Gieser, Linn, Roger, Jérôme E., Perron, Muriel, Institut des Neurosciences Paris-Saclay (NeuroPSI), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), National Institutes of Health [Bethesda] (NIH), Centre d'Etudes et de Recherche Thérapeutique en Ophtalmologie (CERTO), Association RETINA France, and Partenaires INRAE-Partenaires INRAE

Subjects
MESH: Signal Transduction, retina, MESH: Retinal Degeneration, [SDV.NEU.NB]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Neurobiology, Blotting, Western, Ependymoglial Cells, Hippo/YAP pathway, Cell Cycle Proteins, Real-Time Polymerase Chain Reaction, MESH: Phosphoproteins, Mice, MESH: Photoreceptor Cells, MESH: Mice, Inbred C57BL, MESH: Blotting, Western, Animals, MESH: Animals, Photoreceptor Cells, RNA, Messenger, MESH: Mice, MESH: Adaptor Proteins, Signal Transducing, MESH: RNA, Messenger, photoreceptor degeneration, Adaptor Proteins, Signal Transducing, Müller cells, [SDV.NEU.PC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Psychology and behavior, MESH: Real-Time Polymerase Chain Reaction, Retinal Degeneration, [SDV.NEU.SC]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC]/Cognitive Sciences, Nuclear Proteins, TEA Domain Transcription Factors, MESH: Immunohistochemistry, YAP-Signaling Proteins, MESH: Transcription Factors, MESH: Ependymoglial Cells, Phosphoproteins, MESH: Gene Expression Regulation, Immunohistochemistry, DNA-Binding Proteins, Mice, Inbred C57BL, Disease Models, Animal, Retinal Cell Biology, Gene Expression Regulation, sense organs, MESH: Disease Models, Animal, MESH: Nuclear Proteins, MESH: DNA-Binding Proteins, Signal Transduction, Transcription Factors
Abstract

Purpose During retinal degeneration, Müller glia cells respond to photoreceptor loss by undergoing reactive gliosis, with both detrimental and beneficial effects. Increasing our knowledge of the complex molecular response of Müller cells to retinal degeneration is thus essential for the development of new therapeutic strategies. The purpose of this work was to identify new factors involved in Müller cell response to photoreceptor cell death. Methods Whole transcriptome sequencing was performed from wild-type and degenerating rd10 mouse retinas at P30. The changes in mRNA abundance for several differentially expressed genes were assessed by quantitative RT-PCR (RT-qPCR). Protein expression level and retinal cellular localization were determined by western blot and immunohistochemistry, respectively. Results Pathway-level analysis from whole transcriptomic data revealed the Hippo/YAP pathway as one of the main signaling pathways altered in response to photoreceptor degeneration in rd10 retinas. We found that downstream effectors of this pathway, YAP and TEAD1, are specifically expressed in Müller cells and that their expression, at both the mRNA and protein levels, is increased in rd10 reactive Müller glia after the onset of photoreceptor degeneration. The expression of Ctgf and Cyr61, two target genes of the transcriptional YAP/TEAD complex, is also upregulated following photoreceptor loss. Conclusions This work reveals for the first time that YAP and TEAD1, key downstream effectors of the Hippo pathway, are specifically expressed in Müller cells. We also uncovered a deregulation of the expression and activity of Hippo/YAP pathway components in reactive Müller cells under pathologic conditions.

Published
2017

19. Dermatomyosites Nouveaux anticorps, nouvelle classification

Author

Loïs, Bolko, Cyril, Gitiaux, Yves, Allenbach, Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Institut de Myologie, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), CHU Pitié-Salpêtrière [AP-HP], and Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)

Subjects
Adult, MESH: Terminology as Topic, MESH: Interferon-Induced Helicase, IFIH1, Interferon-Induced Helicase, IFIH1, MESH: Age of Onset, [SDV]Life Sciences [q-bio], MESH: Ubiquitin-Activating Enzymes, Ubiquitin-Activating Enzymes, Dermatomyositis, MESH: Dermatomyositis, Terminology as Topic, MESH: Child, MESH: Adenosine Triphosphatases, Humans, MESH: Autoantibodies, Age of Onset, Child, Autoantibodies, Adenosine Triphosphatases, MESH: Humans, MESH: Adult, MESH: Transcription Factors, MESH: Mi-2 Nucleosome Remodeling and Deacetylase Complex, DNA-Binding Proteins, Lung Diseases, Interstitial, MESH: Lung Diseases, Interstitial, MESH: DNA-Binding Proteins, Mi-2 Nucleosome Remodeling and Deacetylase Complex, Transcription Factors
Abstract

Dermatomyositis are rare chronic auto-immune diseases characterized by cutaneous involvement. Diagnosis could be made in childhood or in aldult. There are some different clinical and histological presentation associated with different myositis specific antibody. There are five dermatomyositis specific autoantibodies, anti-Mi2, anti-Tif1-γ, anti-NXP2, anti-MDA5, and anti-SAE. Anti-Mi2 are associated with "classical form" of DM with cutaneous and muscular involvement. Anti-Tif1γ and anti-NXP2 are found in juvenile and adult dermatomyositis, and are associated with recurrent diseases with cutaneous involvement at the forefront. In adults, they are associated with cancer. Anti-MDA5 antibodies are associated with a systemic involvement and an interstitial lung disease. Finally, anti-SAE have been found only in adults, with a classic form.Dermatomyosites Nouveaux anticorps, nouvelle classification.Les dermatomyosites (DM) sont des maladies auto-immunes rares du groupe des myopathies inflammatoires idiopathiques, définies par une atteinte cutanée caractéristique. Elles peuvent survenir dans l’enfance, ou chez l’adulte. Il existe des variations phénotypiques entre les DM concernant la présentation cutanéomusculaire (ex: amyopathique) mais aussi la présentation extra-cutanéomusculaire (ex: atteinte pulmonaire ou articulaire associée). Le caractère auto-immun de ces pathologies est souligné dans 60 % des cas par la présence d’anticorps spécifique de myosite. Ces derniers sont associés à la présence de caractéristiques cliniques, histologiques, mais aussi pronostiques. Ils sont au nombre de cinq, les anti-Mi2, anti-Tif1-γ, anti-NXP2, anti-MDA5 et anti-SAE. Les anti-Mi2 sont associées à une forme clinique cutanée classique, une atteinte musculaire souvent sévère au diagnostic et une bonne évolution sous traitement. Les deux suivants, fréquents chez l’enfant et l’adulte, sont associés à des formes récidivantes cutanées et sont fortement associés aux cancers chez l’adulte. Les anti-MDA5 sont les anticorps associés aux formes les plus systémiques avec une atteinte pulmonaire interstitielle rapidement progressive pouvant être très grave. Enfin, les anti-SAE n’ont été décrits que chez l’adulte, avec une atteinte classique.

Published
2019

20. Condensin-Mediated Chromosome Folding and Internal Telomeres Drive Dicentric Severing by Cytokinesis

Author

Natalja Barinova, Romain Koszul, Stéphane Marcand, Alice Deshayes, Claire Béneut, Karine Dubrana, Virginia Lopez, Agnès Thierry, Thomas Guerin, Luciana Lazar-Stefanita, Stabilité génétique, cellules souches et radiations (SGCSR (U_1274 / UMR_E_008)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Université Paris Cité (UPCité), Régulation spatiale des Génomes - Spatial Regulation of Genomes, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), This work was supported by funding to S.M. from Fondation ARC, EDF, CEA Radiobiology call, DRF-Impulsion (4D-DSB-DIC), and ANR (DICENs-ANR-14-CE10-0021-01), to K.D. from the European Research Council under the Seventh Framework Program (FP7/2007 2013/ERC grant agreement 281287), and to R.K. from the European Research Council under the Horizon 2020 Program (ERC grant agreement 260822). T.M.G. was supported by a PhD fellowship from CEA, ANR, and a Fondation ARC young researcher grant., We thank Angela Taddei for lacI, lacI∗∗, and lacO array plasmids and suggestions, Frank Uhlmann and Thomas Kuilman for the G20 plasmid, Helle Ulrich for the AID tool kit, Didier Busso and Eléa Dizet (CIGEX platform) for the Rap1 sites plasmids, Pascale Lesage for the anti-Dps1 antibody, Rémi Montagne for assistance with the Hi-C data, Romain Le Bars (IMAGE-GIF platform) and Lamya Irbah (IRCM microscopy platform) for assistance with higher-resolution microscopy, Dan Throsby for text editing, and John Marko, Damien D’Amours, Sarah Lambert, François-Xavier Barre, Pablo Radicella, Eric Coïc, Laurent Maloisel, Paul-Henri Roméo, Mathias Toulouze, and Maoussi Lhuillier-Akakpo for fruitful discussions and suggestions., ANR-14-CE10-0021,DICENs,Prévention et résolution des chromosomes dicentriques(2014), European Project: 281287,EC:FP7:ERC,ERC-2011-StG_20101109,NDOGS(2012), European Project: 260822,EC:FP7:ERC,ERC-2010-StG_20091118,DICIG(2011), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Université de Paris (UP), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Condensin, Gene Expression, yeast, Shelterin Complex, MESH: Telomere-Binding Proteins, chemistry.chemical_compound, Chromosome Breakpoints, 0302 clinical medicine, MESH: Saccharomyces cerevisiae Proteins, Hi-C, MESH: Models, Genetic, DNA, Fungal, Adenosine Triphosphatases, 0303 health sciences, biology, SMC, MESH: Karyotype, MESH: Chromosome Breakpoints, MESH: Transcription Factors, Telomere, MESH: Saccharomyces cerevisiae, MESH: Chromosomes, Fungal, Cell biology, DNA-Binding Proteins, Chromosomes, Fungal, mutagenesis, lacI, MESH: Cytokinesis, Saccharomyces cerevisiae Proteins, MESH: Gene Expression, Saccharomyces cerevisiae, Karyotype, Telomere-Binding Proteins, 03 medical and health sciences, Dicentric chromosome, MESH: Adenosine Triphosphatases, Telophase, Molecular Biology, Mitosis, 030304 developmental biology, Cytokinesis, mitosis, Models, Genetic, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, MESH: Multiprotein Complexes, biology.organism_classification, telophase, abscission, MESH: DNA, Fungal, enzymes and coenzymes (carbohydrates), chemistry, Multiprotein Complexes, biology.protein, MESH: Telomere, 030217 neurology & neurosurgery, DNA, MESH: DNA-Binding Proteins, Transcription Factors, condensing
Abstract

International audience; In Saccharomyces cerevisiae, dicentric chromosomes stemming from telomere fusions preferentially break at the fusion. This process restores a normal karyotype and protects chromosomes from the detrimental consequences of accidental fusions. Here, we address the molecular basis of this rescue pathway. We observe that tandem arrays tightly bound by the telomere factor Rap1 or a heterologous high-affinity DNA binding factor are sufficient to establish breakage hotspots, mimicking telomere fusions within dicentrics. We also show that condensins generate forces sufficient to rapidly refold dicentrics prior to breakage by cytokinesis and are essential to the preferential breakage at telomere fusions. Thus, the rescue of fused telomeres results from a condensin- and Rap1-driven chromosome folding that favors fusion entrapment where abscission takes place. Because a close spacing between the DNA-bound Rap1 molecules is essential to this process, Rap1 may act by stalling condensins.

Published
2019

21. Mutations in ACTL6B Cause Neurodevelopmental Deficits and Epilepsy and Lead to Loss of Dendrites in Human Neurons

Author

Jacques L. Michaud, Jessica Sebastian, Hanrong Wu, Dick Lindhout, Karla Manzano-Vargas, Nuwan C. Hettige, Ingrid M. Wentzensen, Huashan Peng, Amy Crunk, Heika Silviera, Malvin Jefri, Henry Houlden, Fadi F. Hamdan, Zahia Aouabed, Stephanie Efthymiou, Xin Zhang, Renske Oegema, Arnaud Tanti, Jerry Vockley, Gustavo Turecki, Julien van Gils, Amber G. Begtrup, Christina Nassif, Gunnar Houge, Naomichi Matsumoto, Thomas Bourgeron, Jean-François Théroux, Emily Fassi, Noriko Miyake, Robert D. Nicholls, Jose E. Martinez, Kristin Herman, Lilit Antonyan, Philippe M. Campeau, Margaret G. Au, Dominic Nelson, Vincenzo Salpietro, Scott C. Bell, Pierre Priam, Joshua L. Deignan, Gregory M. Cooper, Rune Østern, Han G. Brunner, Dagmar Huhle, Amy Goldstein, John M. Graham, Christine Coubes, Koen L.I. van Gassen, Tabib Dabir, Maria Hafström, Simon Gravel, Sophie Ehresmann, Elsa Rossignol, Ilaria Kolobova, Walla Al-Hertani, Julie A. Lessard, Lionel Carmant, Sonja Martin, Richard Delorme, Carl Ernst, Jolien S. Klein Wassink-Ruiter, Naguib Mechawar, Yoshio Makita, Candice R. Finnila, Rami Abou Jamra, Anne Lortie, Justine Rousseau, Sarju G. Mehta, Lina Ghaloul-Gonzalez, MUMC+: DA Klinische Genetica (5), Klinische Genetica, RS: GROW - R4 - Reproductive and Perinatal Medicine, McGill University = Université McGill [Montréal, Canada], Université de Montréal (UdeM), University of Pittsburgh (PITT), Pennsylvania Commonwealth System of Higher Education (PCSHE), GeneDx [Gaithersburg, MD, USA], University of California [Davis] (UC Davis), University of California, David Geffen School of Medicine [Los Angeles], University of California [Los Angeles] (UCLA), University of California-University of California, University of Calgary, UCL, Institute of Neurology [London], Yokohama City University (YCU), Asahikawa Medical College, Trondheim University, Haukeland University Hospital, University of Bergen (UiB), Washington University School of Medicine in St. Louis, Washington University in Saint Louis (WUSTL), University of Groningen [Groningen], University Medical Center Groningen [Groningen] (UMCG), Children's Hospital of Pittsburgh of UPMC [Etats-Unis], Belfast City Hospital, Génétique humaine et fonctions cognitives - Human Genetics and Cognitive Functions (GHFC (UMR_3571 / U-Pasteur_1)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Child and Adolescent Psychiatry Department [AP- HP Hôpital Robert Debré], AP-HP Hôpital universitaire Robert-Debré [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), HudsonAlpha Institute for Biotechnology [Huntsville, AL], Children’s Rehabilitation Service [Mobile, AL] (CRS), Children's Rehabilitation Service [Montgomery, AL] (CRS), University Medical Center [Utrecht], Cambridge University Hospitals - NHS (CUH), University of Cambridge [UK] (CAM), University Hospital Leipzig, Donders Institute for Brain, Cognition and Behaviour, Radboud university [Nijmegen], Maastricht University Medical Centre (MUMC), Maastricht University [Maastricht], Cedars-Sinai Medical Center, CHU Montpellier, Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), We acknowledge funding by FRQS doctoral (S.B.), Indonesian Endowment Fund for Education PhD award (M.J.), CONACYT (Mexico) and MITACS (K.M.V.), Genome Canada and Génome Québec (J.L.M. and E.R.), Canada Research Chairs program (G.T. and C.E.), AMED, MEXT, JST, MHLW, and Takeda Science Foundation (N. Matsumoto), and CIHR grant (C.E. and P.M.C.)., University of California (UC), University of California (UC)-University of California (UC), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Radboud University [Nijmegen], and Faculteit Medische Wetenschappen/UMCG

Subjects
0301 basic medicine, Male, PROTEIN EXPRESSION, Chromosomal Proteins, Non-Histone, Mutant, MESH: Neurons, 0302 clinical medicine, SYNAPTIC PLASTICITY, MESH: Child, Genetics(clinical), Global developmental delay, TRANSCRIPTION, Child, Genetics (clinical), ACTL6B, genetic engineering, intellectual disability, neurodevelopment, seizure, stem cells, Genetics, Neurons, SWI/SNF COMPLEX, MESH: Infant, Hypotonia, Neural stem cell, Chromatin, DNA-Binding Proteins, medicine.anatomical_structure, MESH: Young Adult, MESH: Epilepsy, Child, Preschool, Female, medicine.symptom, Adult, MESH: Mutation, DISORDERS, Induced Pluripotent Stem Cells, Biology, MESH: Induced Pluripotent Stem Cells, MESH: Actins, MESH: Dendrites, Chromatin remodeling, Article, MESH: Chromatin, 03 medical and health sciences, Young Adult, All institutes and research themes of the Radboud University Medical Center, MESH: Chromosomal Proteins, Non-Histone, medicine, Journal Article, Humans, CHROMATIN REMODELING COMPLEX, Progenitor cell, Loss function, MESH: Neurodevelopmental Disorders, MESH: Humans, Epilepsy, Neurodevelopmental disorders Donders Center for Medical Neuroscience [Radboudumc 7], [SCCO.NEUR]Cognitive science/Neuroscience, MESH: Child, Preschool, COFFIN-SIRIS SYNDROME, MEMORY, Infant, MESH: Adult, Dendrites, GENE, MESH: Male, Actins, 030104 developmental biology, Neurodevelopmental Disorders, Mutation, OLIGODENDROCYTE, Neuron, MESH: Female, 030217 neurology & neurosurgery, MESH: DNA-Binding Proteins
Abstract

International audience; We identified individuals with variations in ACTL6B, a component of the chromatin remodeling machinery including the BAF complex. Ten individuals harbored bi-allelic mutations and presented with global developmental delay, epileptic encephalopathy, and spasticity, and ten individuals with de novo heterozygous mutations displayed intellectual disability, ambulation deficits, severe language impairment, hypotonia, Rett-like stereotypies, and minor facial dysmorphisms (wide mouth, diastema, bulbous nose). Nine of these ten unrelated individuals had the identical de novo c.1027G>A (p.Gly343Arg) mutation. Human-derived neurons were generated that recaptured ACTL6B expression patterns in development from progenitor cell to post-mitotic neuron, validating the use of this model. Engineered knock-out of ACTL6B in wild-type human neurons resulted in profound deficits in dendrite development, a result recapitulated in two individuals with different bi-allelic mutations, and reversed on clonal genetic repair or exogenous expression of ACTL6B. Whole-transcriptome analyses and whole-genomic profiling of the BAF complex in wild-type and bi-allelic mutant ACTL6B neural progenitor cells and neurons revealed increased genomic binding of the BAF complex in ACTL6B mutants, with corresponding transcriptional changes in several genes including TPPP and FSCN1, suggesting that altered regulation of some cytoskeletal genes contribute to altered dendrite development. Assessment of bi-alleic and heterozygous ACTL6B mutations on an ACTL6B knock-out human background demonstrated that bi-allelic mutations mimic engineered deletion deficits while heterozygous mutations do not, suggesting that the former are loss of function and the latter are gain of function. These results reveal a role for ACTL6B in neurodevelopment and implicate another component of chromatin remodeling machinery in brain disease.

Published
2019

22. Regulatory control of DNA end resection by Sae2 phosphorylation

Author

Cannavo, Elda, Johnson, Dominic, Andres, Sara, Kissling, Vera, Reinert, Julia, Garcia, Valerie, Erie, Dorothy, Hess, Daniel, Thomä, Nicolas, Enchev, Radoslav, Peter, Matthias, Williams, R Scott, Neale, Matt, Cejka, Petr, Williams, R. Scott, Friedrich Miescher Institute for Biomedical Research (FMI), Novartis Research Foundation, Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Institute of Biochemistry, Universität Zürich [Zürich] = University of Zurich (UZH), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, and Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
DNA End-Joining Repair, Saccharomyces cerevisiae Proteins, [SDV]Life Sciences [q-bio], MESH: DNA Breaks, Double-Stranded, MESH: Exodeoxyribonucleases, MESH: Cell Cycle, Saccharomyces cerevisiae, Article, MESH: Saccharomyces cerevisiae Proteins, MESH: Endonucleases, MESH: Protein Binding, DNA Breaks, Double-Stranded, MESH: Endodeoxyribonucleases, Phosphorylation, DNA, Fungal, Endodeoxyribonucleases, MESH: Phosphorylation, MESH: Protein Multimerization, Cell Cycle, Recombinational DNA Repair, MESH: DNA End-Joining Repair, MESH: Multiprotein Complexes, Endonucleases, MESH: Saccharomyces cerevisiae, DNA-Binding Proteins, MESH: Recombinational DNA Repair, MESH: DNA, Fungal, Meiosis, enzymes and coenzymes (carbohydrates), MESH: Meiosis, Exodeoxyribonucleases, Multiprotein Complexes, Protein Multimerization, MESH: DNA-Binding Proteins, Protein Binding
Abstract

DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Resected DNA ends are refractory to end-joining mechanisms and are instead channeled to homology-directed repair. Using biochemical, genetic, and imaging methods, we show that phosphorylation of Saccharomyces cerevisiae Sae2 controls its capacity to promote the Mre11-Rad50-Xrs2 (MRX) nuclease to initiate resection of blocked DNA ends by at least two distinct mechanisms. First, DNA damage and cell cycle-dependent phosphorylation leads to Sae2 tetramerization. Second, and independently, phosphorylation of the conserved C-terminal domain of Sae2 is a prerequisite for its physical interaction with Rad50, which is also crucial to promote the MRX endonuclease. The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and therefore the choice between the key DNA double-strand break repair mechanisms., It has previously been established that DNA end resection in yeast and in humans is under CDK control. Here the authors explain how phosphorylation regulates the capacity of Sae2 — the yeast orthologue of human CtIP — to promote DNA end resection.

Published
2018

23. Tetramerization and interdomain flexibility of the replication initiation controller YabA enables simultaneous binding to multiple partners

Author

Liza Felicori, Magali Ventroux, Transito Garcia-Garcia, Laurent Terradot, Franck Molina, Anthony J. Wilkinson, Mark J. Fogg, Philippe Noirot, Katie H. Jameson, Alexandre Bazin, Mickaël V. Cherrier, Marie Francoise Noirot-Gros, Pierre Roblin, Sysdiag CNRS Bio-Rad UMR 3145, Cap Delta/Parc Euromédecine, Centre National de la Recherche Scientifique (CNRS), Département Caractérisation et Elaboration des Produits Issus de l'Agriculture (CEPIA), Institut National de la Recherche Agronomique (INRA), York Structural Biology Laboratory, Department of Chemistry, University of York [York, UK], MICrobiologie de l'ALImentation au Service de la Santé (MICALIS), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Informatique, de Modélisation et d'Optimisation des Systèmes (LIMOS), Ecole Nationale Supérieure des Mines de St Etienne-Université Clermont Auvergne (UCA)-Centre National de la Recherche Scientifique (CNRS), Sysdiag-Modélisation et Ingénierie des Systèmes Complexes Biologiques pour le Diagnostic (SysDiag ), BIO-RAD-Centre National de la Recherche Scientifique (CNRS), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Ecole Nationale Supérieure des Mines de St Etienne-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), European integrated project, BaSysBio, Biotechnology and Biological Sciences Research Council, UK, EU-Marie Curie Project AMBER FP7-People [317338], CIBLE program from the region Rhones-alpes, MICALIS INRA, Felicori, Liza, Jameson, Katie H., Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), and Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry (MMSB)

Subjects
Models, Molecular, 0301 basic medicine, Protein Conformation, Amino Acid Motifs, Intracellular Space, dnaN, MESH: DNA Replication, Plasma protein binding, MESH: Amino Acid Sequence, MESH: Zinc, MESH: Amino Acid Motifs, Protein structure, MESH: Structure-Activity Relationship, MESH: Protein Conformation, Structural Biology, Protein Interaction Mapping, MESH: Bacterial Proteins, Genetics, DNA clamp, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Protein Multimerization, DNA-Binding Proteins, Protein Transport, Zinc, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Intracellular Space, MESH: Models, Molecular, Bacillus subtilis, Protein Binding, DNA Replication, MESH: Protein Transport, MESH: Mutation, Molecular Sequence Data, MESH: Sequence Alignment, Biology, Structure-Activity Relationship, 03 medical and health sciences, Bacterial Proteins, Position-Specific Scoring Matrices, MESH: Protein Binding, Protein Interaction Domains and Motifs, Amino Acid Sequence, Homology modeling, MESH: Protein Interaction Domains and Motifs, Binding Sites, MESH: Molecular Sequence Data, MESH: Protein Interaction Mapping, DNA replication, MESH: Position-Specific Scoring Matrices, MESH: Bacillus subtilis, MESH: Multiprotein Complexes, DnaA, 030104 developmental biology, MESH: Binding Sites, Replication Initiation, Multiprotein Complexes, Mutation, Biophysics, Protein Multimerization, Sequence Alignment, MESH: DNA-Binding Proteins
Abstract

International audience; YabA negatively regulates initiation of DNA replication in low-GC Gram-positive bacteria. The protein exerts its control through interactions with the initiator protein DnaA and the sliding clamp DnaN. Here, we combined X-ray crystallography, X-ray scattering (SAXS), modeling and biophysical approaches, with in vivo experimental data to gain insight into YabA function. The crystal structure of the N-terminal domain (NTD) of YabA solved at 2.7 Å resolution reveals an extended α-helix that contributes to an intermolecular four-helix bundle. Homology modeling and biochemical analysis indicates that the C-terminal domain (CTD) of YabA is a small Zn-binding domain. Multi-angle light scattering and SAXS demonstrate that YabA is a tetramer in which the CTDs are independent and connected to the N-terminal four-helix bundle via flexible linkers. While YabA can simultaneously interact with both DnaA and DnaN, we found that an isolated CTD can bind to either DnaA or DnaN, individually. Site-directed mutagenesis and yeast-two hybrid assays identified DnaA and DnaN binding sites on the YabA CTD that partially overlap and point to a mutually exclusive mode of interaction. Our study defines YabA as a novel structural hub and explains how the protein tetramer uses independent CTDs to bind multiple partners to orchestrate replication initiation in the bacterial cell.

Published
2016

24. High prevalence of abnormal cervical smears in a hospital cohort of French women beyond the upper age limit screening program

Author

Christiane Mougin, Isabelle Bedgedjian, Essaada Belglaiaa, Alexandra Luquain, Jean-Luc Prétet, Didier Riethmuller, Said Chouham, Sindy Vrecko, Séverine Valmary-Degano, David Guenat, Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon), Carcinogénèse épithéliale : facteurs prédictifs et pronostiques - UFC (EA 3181) (CEF2P / CARCINO), Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Interactions hôte-greffon-tumeur, ingénierie cellulaire et génique - UFC (UMR INSERM 1098) (RIGHT), Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang [Bourgogne-Franche-Comté] (EFS [Bourgogne-Franche-Comté])-Université de Franche-Comté (UFC), and Service d'Anatomie pathologique [CHRU Besançon]

Subjects
HPV 16, MESH: Human Papillomavirus DNA Tests, Epidemiology, hrHPV, Uterine Cervical Neoplasms, MESH: Hospitalization, MESH: Papillomavirus Infections, Human Papillomavirus DNA Tests, MESH: Genotype, MESH: Aged, 80 and over, 0302 clinical medicine, MESH: Papillomaviridae, Pap smears, Uterine Cervical Dysplasia, Prevalence, Mass Screening, 030212 general & internal medicine, Papillomaviridae, Human Papillomavirus DNA Test, Early Detection of Cancer, MESH: Aged, Aged, 80 and over, Cervical cancer, education.field_of_study, biology, Obstetrics, 3. Good health, MESH: Uterine Cervical Neoplasms, DNA-Binding Proteins, Hospitalization, 030220 oncology & carcinogenesis, Cohort, Female, France, Papanicolaou Test, medicine.medical_specialty, Genotype, MESH: Uterine Cervical Dysplasia, Population, [SDV.CAN]Life Sciences [q-bio]/Cancer, Women aged over 65, MESH: Papanicolaou Test, 03 medical and health sciences, medicine, MESH: Early Detection of Cancer, Humans, MESH: Mass Screening, education, MESH: Prevalence, Mass screening, Aged, Retrospective Studies, Gynecology, MESH: Humans, business.industry, Papillomavirus Infections, Public Health, Environmental and Occupational Health, MESH: Retrospective Studies, Retrospective cohort study, medicine.disease, biology.organism_classification, MESH: France, business, MESH: Female, MESH: DNA-Binding Proteins
Abstract

Objective To determine the prevalence of cytological abnormalities and high risk Human PapillomaVirus (hrHPV) in cervical smears from French women aged over 65 years who attended the referent Gynecology Clinic of the Besancon University Hospital. Methods Between 2002 and 2012, 796 French women aged 66–99 years were cotested for cytology and hrHPV by Hybrid Capture 2 (hc2). hc2-positive cases were subjected to real time PCR for specific HPV 16/18/45 genotyping. Women with normal Pap smears and positive for hrHPV were followed-up every 12 months. Results Cytological abnormalities were detected in more than 30% of women and cervical cancers (CC) in 2.9% of women. Benign lesions were more frequent in women aged 66–75 years while (pre)-malignant lesions were preferentially found in women over 76. The prevalence of hrHPV was 22.7%. HPV 16 was the most frequent (23.8%), followed by HPV 45 (7.7%) and HPV 18 (3.9%). The rate of hrHPV increased with the lesion severity and HPV 16 was identified in 50% of CC. Among the followed-up women, those who developed CIN3 were HPV16 positive at study entry. Conclusion The study provides important estimates of the prevalence of cervical abnormalities and hrHPV positivity in a French hospital based-population over 65. Findings suggest to consider this high risk population in regards to cervical cancer.

Published
2015

25. Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells

Author

Stephen P. Jackson, Mareike Herzog, Alejandra Bruna, Luca Pellegrini, Violeta Serra, Mark J. O'Connor, Zhongwu Lai, Chloé Lescale, Jacqueline J.L. Jacobs, Fengtang Yang, Jonathan Lam, Matylda Sczaniecka-Clift, Abigail Shea, Carlos Caldas, Matthias Ostermaier, Gabriel Balmus, Julia Coates, Wenming Wei, Inge de Krijger, Yaron Galanty, Mukerrem Demir, Ludovic Deriano, Petra Beli, Domenic Pilger, Harveer Dev, Rimma Belotserkovskaya, Alistair Martin, Beiyuan Fu, Ting-Wei Will Chiang, Qian Wu, Dev, Harveer [0000-0003-2874-6894], Yang, Fengtang [0000-0002-3573-2354], Balmus, Gabriel [0000-0003-2872-4468], Serra, Violeta [0000-0001-6620-1065], Beli, Petra [0000-0001-9507-9820], Pellegrini, Luca [0000-0002-9300-497X], Deriano, Ludovic [0000-0002-9673-9525], Jacobs, Jacqueline JL [0000-0002-7704-4795], Jackson, Stephen P [0000-0001-9317-7937], Apollo - University of Cambridge Repository, Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge [UK] (CAM), Department of Biochemistry [Cambridge], Cambridge University Hospitals - NHS (CUH), Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris] (IP), Netherlands Cancer Institute (NKI), Antoni van Leeuwenhoek Hospital, Cancer Research UK Cambridge Institute [Cambridge, Royaume-Uni] (CRUK), Institute of Molecular Biology (IMB), Johannes Gutenberg - Universität Mainz = Johannes Gutenberg University (JGU), Wellcome Trust Sanger Institute [Hinxton, UK], AstraZeneca US [Waltham, USA], AstraZeneca [Cambridge, UK], The Wellcome Trust Sanger Institute [Cambridge], Vall d'Hebron Institute of Oncology [Barcelone] (VHIO), Vall d'Hebron University Hospital [Barcelona], The SPJ lab is largely funded by a Cancer Research UK (CRUK) Program Grant, C6/A18796, and a Wellcome Trust (WT) Investigator Award, 206388/Z/17/Z. Core infrastructure funding was provided by CRUK grant C6946/A24843 and WT grant WT203144. S.P.J. receives a salary from the University of Cambridge. H.D. is funded by WT Clinical Fellowship 206721/Z/17/Z. TWC was supported by a Cambridge International Scholarship. D.P. is funded by Cancer Research UK studentship C6/A21454. The P.B. lab is supported by the Emmy Noether Program (BE 5342/1-1) from the German Research Foundation and a Marie Curie Career Integration Grant from the European Commission (630763). The L.P. lab is funded by the WT (investigator award 104641/Z/14/Z) and the Medical Research Council (project grant MR/N000161/1). The C.C. lab was supported with funding from CRUK. The J.J. lab was supported by the European Research Council grant ERC-StG 311565, The Dutch Cancer Society (KWF) grant KWF 10999, and the Netherlands Organization for Scientific Research (NWO) as part of the National Roadmap Large-scale Research Facilities of the Netherlands, Proteins@Work (project no. 184.032.201 to the Proteomics Facility of the Netherlands Cancer Institute). The L.D. lab is funded by the Institut Pasteur, the Institut National du Cancer (no. PLBIO16-181) and the European Research Council (starting grant agreement no. 310917). W.W. is part of the Pasteur–Paris University (PPU) International PhD program and this project received funding from the CNBG company, China. Q.W. is funded by the Wellcome Trust (200814/Z/16/Z ). The V.S. lab work was funded by the Instituto de Salud Carlos III (ISCIII), an initiative of the Spanish Ministry of Economy and Innovation partially supported by European Regional Development FEDER Funds (PI17-01080 to VS), the European Research Area-NET, Transcan-2 (AC15/00063), a non-commercial research agreement with AstraZeneca UK, and structural funds from the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR, 2017 SGR 540) and the Orozco Family. V.S. received a salary and travel support to C.C.’s lab from ISCIII (CP14/00228, MV15/00041) and the FERO Foundation., The authors thank all S.P.J. laboratory members for support and advice, and Cambridge colleagues N. Lawrence for OMX super-resolution microscopy support and R. Butler for help with computational image analyses and programming. The authors also thank S. Selivanova and S. Hough for help with plasmid amplification, sample preparation and tissue culture maintenance, K. Dry for extensive editorial assistance, F. Muñoz-Martinez for assistance with CRISPR–Cas9 knockout generation, L. Radu for assistance with protein purification, C. Lord (Institute of Cancer Research, London) for SUM149PT cells, D. Durocher (University of Toronto, Canada) for U2OS LacSceIII cells, F. Alt (Harvard University, USA) for CH12F3 cells and 53bp1 knockout CH12F3 cell clones, T. Honjo (Kyoto University, Japan) for permission to use the CH12F3 cell line, and J. Serrat in the Jacobs lab for technical assistance, Institut Pasteur [Paris], and Johannes Gutenberg - Universität Mainz (JGU)

Subjects
MESH: DNA Breaks, Double-Stranded, RAD51, Cell Cycle Proteins, Poly (ADP-Ribose) Polymerase Inhibitor, MESH: Recombinational DNA Repair, Mice, MESH: Animals, DNA Breaks, Double-Stranded, skin and connective tissue diseases, Cancer, Telomere-binding protein, Ovarian Neoplasms, MESH: Breast Neoplasms / metabolism, MESH: Telomere-Binding Proteins / metabolism, 3. Good health, Cell biology, MESH: HEK293 Cells, MESH: Proteins / genetics, MESH: Telomere-Binding Proteins / genetics, MESH: Tumor Suppressor p53-Binding Protein 1 / metabolism, MESH: Xenograft Model Antitumor Assays, Telomere-Binding Proteins, MESH: Ovarian Neoplasms / drug therapy, Bone Neoplasms, MESH: Ovarian Neoplasms / metabolism, Article, 03 medical and health sciences, MESH: Cell Cycle Proteins, MESH: Bone Neoplasms / metabolism, Humans, MESH: Osteosarcoma / metabolism, [SDV.GEN]Life Sciences [q-bio]/Genetics, MESH: Humans, MESH: Tumor Suppressor p53-Binding Protein 1 / genetics, Dose-Response Relationship, Drug, HEK 293 cells, Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, DNA, MESH: BRCA1 Protein / deficiency, 030104 developmental biology, Multiprotein Complexes, MESH: Mad2 Proteins / metabolism, MESH: Breast Neoplasms / genetics, MESH: Bone Neoplasms / drug therapy, Cisplatin, Homologous recombination, MESH: Osteosarcoma / genetics, MESH: Female, 0301 basic medicine, DNA End-Joining Repair, MESH: Proteins / metabolism, MESH: Dose-Response Relationship, Drug, chemistry.chemical_compound, MESH: Osteosarcoma / pathology, MESH: Breast Neoplasms / pathology, Homologous Recombination, Polymerase, MESH: Breast Neoplasms / drug therapy, Osteosarcoma, biology, Chemistry, BRCA1 Protein, DNA damage and repair, MESH: Poly(ADP-ribose) Polymerase Inhibitors / pharmacology, MESH: Bone Neoplasms / genetics, DNA-Binding Proteins, MESH: Bone Neoplasms / pathology, Mad2 Proteins, Female, MESH: Ovarian Neoplasms / genetics, Tumor Suppressor p53-Binding Protein 1, MESH: Cisplatin / pharmacology, MESH: Cell Line, Tumor, Lymphocytes, Null, [SDV.CAN]Life Sciences [q-bio]/Cancer, Breast Neoplasms, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: BRCA1 Protein / genetics, Poly(ADP-ribose) Polymerase Inhibitors, Cell Line, Tumor, MESH: Drug Resistance, Neoplasm* / genetics, MESH: Mad2 Proteins / genetics, MESH: Ovarian Neoplasms / pathology, Animals, MESH: Mice, MESH: Osteosarcoma / drug therapy, Oligonucleotide, Protective Devices, Recombinational DNA Repair, Cell Biology, MESH: Multiprotein Complexes, Xenograft Model Antitumor Assays, HEK293 Cells, Drug Resistance, Neoplasm, biology.protein, MESH: DNA End-Joining Repair, MESH: DNA-Binding Proteins
Abstract

International audience; BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.

Published
2018

26. Mutations in the BAF-complex subunit DPF2 associated with Coffin-Siris syndrome

Author

Andrew O.M. Wilkie, Georgia Vasileiou, Bernt Popp, Yun Li, Maren Wenzel, Marion Gérard, Susan Tomkins, Jenny Morton, Megan T. Cho, André Reis, Astrid Weber, George E. Hoganson, Beate Albrecht, Felix B. Engel, Arif B. Ekici, Maria-Renée Plona, Janine Altmüller, Christian Thiel, Christian Büttner, Jill Clayton-Smith, Karen Low, Dagmar Wieczorek, Deciphering Developmental Disorders Study, Bernd Wollnik, Eduardo Calpena, Nuria C. Bramswig, Sabine Endele, Hermann-Josef Lüdecke, Silvia Vergarajauregui, Tim M. Strom, Institute of Human Genetics, University Erlangen-Nuremberg, Friedrich-Alexander Universität Erlangen-Nürnberg (FAU), Service de Génétique [CHU Caen], Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Normandie Université (NU)-CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Tumorothèque de Caen Basse-Normandie (TCBN), Biologie, génétique et thérapies ostéoarticulaires et respiratoires (BIOTARGEN), Normandie Université (NU)-Normandie Université (NU), Institut für Humangenetik, Regional Genetic Service, St Mary's Hospital, Manchester, West Midlands Regional Genetics Laboratory and Clinical Genetics Unit, Birmingham Women's Hospital, Max Planck Institute for Plant Breeding Research (MPIPZ), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), University Medicine Goettingen, Department of Pediatrics [Chicago, IL, USA] (College of Medicine), University of Illinois [Chicago] (UIC), University of Illinois System-University of Illinois System, GeneDx [Gaithersburg, MD, USA], JRC Institute for Energy and Transport (IET), European Commission - Joint Research Centre [Petten], Institut für Humangenetik [Essen], Universitätsklinikum Essen, Technical University of Munich (TUM), Institute of Human Genetics, University Hospital Erlangen, Institute of Human Genetics [Erlangen, Allemagne], Université de Lorraine (UL), Institute of Human Genetics [Cologne], Universitätsklinikum Köln (Uniklinik Köln)-University of Cologne, University Medical Center Göttingen (UMG), Institute of Human Genetics - Institut für Humangenetik [Essen], Universitätsklinikum Essen [Universität Duisburg-Essen] (Uniklinik Essen)-Universitat Duisberg-Essen, Technische Universität München [München] (TUM)-German Research Center for Environmental Health-Helmholtz-Zentrum München (HZM), CHU Caen, Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Normandie Université (NU)-Tumorothèque de Caen Basse-Normandie (TCBN)-Université de Caen Normandie (UNICAEN), Normandie Université (NU), Universitätsklinikum Essen [Universität Duisburg-Essen] (Uniklinik Essen), Manchester Academic Health Science Centre (MAHSC), University of Manchester [Manchester], Birmingham Women's and Children's NHS Foundation Trust, University Hospitals Bristol, Liverpool Women's NHS Foundation Trust, Genetikum, Cologne Center for Genomics [Cologne] (CCG), University of Cologne, Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), University of Illinois College of Medicine, University of Illinois System, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), and University of Oxford [Oxford]

Subjects
0301 basic medicine, Male, Medizin, PHD finger, MESH: Amino Acid Sequence, Histones, 0302 clinical medicine, MESH: Child, Chlorocebus aethiops, MESH: Hand Deformities, Congenital, Missense mutation, Coffin-Siris syndrome, histone modification, MESH: Animals, BAF complex, nuclear aggregates, Child, Genetics (clinical), Genetics, MESH: Histones, MESH: Protein Subunits, DNA-Binding Proteins, MESH: COS Cells, Histone, Phenotype, DPF2, MESH: Facies, intellectual disability, Child, Preschool, MESH: HEK293 Cells, COS Cells, Female, MESH: Neck, Hand Deformities, Congenital, MESH: Face, MESH: Abnormalities, Multiple, MESH: Mutation, Adolescent, Protein subunit, Micrognathism, dominant negative, autism spectrum disorder, Biology, MESH: Micrognathism, MESH: Phenotype, Frameshift mutation, MESH: Intellectual Disability, 03 medical and health sciences, Report, medicine, Animals, Humans, Abnormalities, Multiple, Amino Acid Sequence, Gene, Coffin–Siris syndrome, MESH: Adolescent, [SDV.GEN]Life Sciences [q-bio]/Genetics, MESH: Humans, Coarse facial features, MESH: Child, Preschool, Facies, medicine.disease, MESH: Cercopithecus aethiops, MESH: Male, Protein Subunits, Autism Spectrum Disorder, Baf Complex, Coffin-siris Syndrome, Dominant Negative, Dpf2, Histone Modification, Intellectual Disability, Nail Hypoplasia, Nuclear Aggregates, Phd Finger, 030104 developmental biology, HEK293 Cells, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, Face, Mutation, biology.protein, nail hypoplasia, MESH: Female, 030217 neurology & neurosurgery, Neck, MESH: DNA-Binding Proteins, Transcription Factors
Abstract

International audience; Variants affecting the function of different subunits of the BAF chromatin-remodelling complex lead to various neurodevelopmental syndromes, including Coffin-Siris syndrome. Furthermore, variants in proteins containing PHD fingers, motifs recognizing specific histone tail modifications, have been associated with several neurological and developmental-delay disorders. Here, we report eight heterozygous de novo variants (one frameshift, two splice site, and five missense) in the gene encoding the BAF complex subunit double plant homeodomain finger 2 (DPF2). Affected individuals share common clinical features described in individuals with Coffin-Siris syndrome, including coarse facial features, global developmental delay, intellectual disability, speech impairment, and hypoplasia of fingernails and toenails. All variants occur within the highly conserved PHD1 and PHD2 motifs. Moreover, missense variants are situated close to zinc binding sites and are predicted to disrupt these sites. Pull-down assays of recombinant proteins and histone peptides revealed that a subset of the identified missense variants abolish or impaire DPF2 binding to unmodified and modified H3 histone tails. These results suggest an impairment of PHD finger structural integrity and cohesion and most likely an aberrant recognition of histone modifications. Furthermore, the overexpression of these variants in HEK293 and COS7 cell lines was associated with the formation of nuclear aggregates and the recruitment of both wild-type DPF2 and BRG1 to these aggregates. Expression analysis of truncating variants found in the affected individuals indicated that the aberrant transcripts escape nonsense-mediated decay. Altogether, we provide compelling evidence that de novo variants in DPF2 cause Coffin-Siris syndrome and propose a dominant-negative mechanism of pathogenicity.

Published
2018

27. Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins

Author

Frédéric Boccard, Axel Cournac, Virginia S. Lioy, Romain Koszul, Olivier Espéli, Stéphane Duigou, Martial Marbouty, Julien Mozziconacci, Conformation et Ségrégation du chromosome bactérien (OCB), Département Biologie des Génomes (DBG), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Régulation spatiale des Génomes - Spatial Regulation of Genomes, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physique Théorique de la Matière Condensée (LPTMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Centre interdisciplinaire de recherche en biologie (CIRB), Labex MemoLife, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Collège de France (CdF (institution))-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), This research was supported by funding from the European Research Council under the 7th Framework Program (FP7/2007-2013, ERC Grant Agreement 260822 to R.K.), by the Agence Nationale pour la Recherche (HiResBac ANR-15-CE11-0023-03 to R.K., O.E., and J.M.), by the Fondation pour la Recherche Médicale (to V.S.L.), and by the Agence Nationale pour la Recherche (ANR-12-BSV8-0020-01 to F.B.)., ANR-15-CE11-0023,HiResBaCS,Structure des chromosomes bactériens revélée a haute résolution(2015), European Project: 260822,EC:FP7:ERC,ERC-2010-StG_20091118,DICIG(2011), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris)

Subjects
0301 basic medicine, MESH: Chromosomes, Bacterial, MESH: Protein Structure, Quaternary, Chromosomal Proteins, Non-Histone, DBG, Condensin, [SDV]Life Sciences [q-bio], MESH: Escherichia coli Proteins, Bacterial genome size, Origin of replication, Genome, General Biochemistry, Genetics and Molecular Biology, MESH: Escherichia coli K12, 03 medical and health sciences, 0302 clinical medicine, HU, Hi-C, MESH: Chromosomal Proteins, Non-Histone, MESH: Adenosine Triphosphatases, Nucleoid, genome organization, Protein Structure, Quaternary, bacteria, Genetics, Adenosine Triphosphatases, OCB, biology, Escherichia coli K12, SMC, Circular bacterial chromosome, Escherichia coli Proteins, Chromosome, Chromosomes, Bacterial, MESH: Multiprotein Complexes, MatP, DNA-Binding Proteins, Repressor Proteins, 030104 developmental biology, MESH: Repressor Proteins, Multiprotein Complexes, biology.protein, chromatin, Nucleoid organization, 030217 neurology & neurosurgery, MESH: DNA-Binding Proteins
Abstract

International audience; As in eukaryotes, bacterial genomes are not randomly folded. Bacterial genetic information is generally carried on a circular chromosome with a single origin of replication from which two replication forks proceed bidirectionally toward the opposite terminus region. Here, we investigate the higher-order architecture of the Escherichia coli genome, showing its partition into two structurally distinct entities by a complex and intertwined network of contacts: the replication terminus (ter) region and the rest of the chromosome. Outside of ter, the condensin MukBEF and the ubiquitous nucleoid-associated protein (NAP) HU promote DNA contacts in the megabase range. Within ter, the MatP protein prevents MukBEF activity, and contacts are restricted to ∼280 kb, creating a domain with distinct structural properties. We also show how other NAPs contribute to nucleoid organization, such as H-NS, which restricts short-range interactions. Combined, these results reveal the contributions of major evolutionarily conserved proteins in a bacterial chromosome organization.

Published
2018

28. Dysregulation of chromatin remodelling complexes in amyotrophic lateral sclerosis

Author

Ekaterina Rogaeva, Julia Keith, Janice Robertson, Heather D. Durham, Benoit J. Gentil, Christine Marques, Beibei Zhao, Caroline Rouaux, Lorne Zinman, Sandra Minotti, Michael Tibshirani, McGill University = Université McGill [Montréal, Canada], University of Toronto, Mécanismes Centraux et Périphériques de la Neurodégénérescence, Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Dieterle, Stéphane

Subjects
0301 basic medicine, Cytoplasm, [SDV]Life Sciences [q-bio], Mutant, MESH: Neurons, MESH: DNA Helicases, medicine.disease_cause, MESH: Spinal Cord, Mice, 0302 clinical medicine, MESH: Animals, Amyotrophic lateral sclerosis, Genetics (clinical), MESH: Amyotrophic Lateral Sclerosis, Motor Neurons, Neurons, Mutation, MESH: RNA-Binding Protein FUS, Nuclear Proteins, Cell Differentiation, General Medicine, MESH: Transcription Factors, MESH: Protein Subunits, Cell biology, DNA-Binding Proteins, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Spinal Cord, MESH: Motor Neurons, MESH: Cell Differentiation, MESH: Mutation, Protein subunit, Biology, 03 medical and health sciences, Genetics, medicine, Animals, Humans, Molecular Biology, MESH: Mice, Messenger RNA, MESH: Humans, MESH: Cytoplasm, Amyotrophic Lateral Sclerosis, DNA Helicases, MESH: Chromatin Assembly and Disassembly, Motor neuron, Chromatin Assembly and Disassembly, Spinal cord, medicine.disease, Protein Subunits, 030104 developmental biology, RNA-Binding Protein FUS, MESH: Nuclear Proteins, 030217 neurology & neurosurgery, MESH: DNA-Binding Proteins, Transcription Factors
Abstract

International audience; Amyotrophic lateral sclerosis is a fatal neurodegenerative disease with paralysis resulting from dysfunction and loss of motor neurons. A common neuropathological finding is attrition of motor neuron dendrites, which make central connections vital to motor control. The chromatin remodelling complex, neuronal Brahma-related gene 1 (Brg1)-associated factor complex (nBAF), is critical for neuronal differentiation, dendritic extension and synaptic function. We have identified loss of the crucial nBAF subunits Brg1, Brg1-associated factor 53b and calcium responsive transactivator in cultured motor neurons expressing FUS or TAR-DNA Binding Protein 43 (TDP-43) mutants linked to familial ALS. When plasmids encoding wild-type or mutant human FUS or TDP-43 were expressed in motor neurons of dissociated spinal cord cultures prepared from E13 mice, mutant proteins in particular accumulated in the cytoplasm. Immunolabelling of nBAF subunits was reduced in proportion to loss of nuclear FUS or TDP-43 and depletion of Brg1 was associated with nuclear retention of Brg1 mRNA. Dendritic attrition (loss of intermediate and terminal dendritic branches) occurred in motor neurons expressing mutant, but not wild-type, FUS or TDP-43. This attrition was delayed by ectopic over-expression of Brg1 and was reproduced by inhibiting Brg1 activity either through genetic manipulation or treatment with the chemical inhibitor, (E)-1-(2-Hydroxyphenyl)-3-((1R, 4R)-5-(pyridin-2-yl)-2, 5-diazabicyclo[2.2.1]heptan-2-yl)prop-2-en-1-one, demonstrating the importance of Brg1 to maintenance of dendritic architecture. Loss of nBAF subunits was also documented in spinal motor neurons in autopsy tissue from familial amyotrophic sclerosis (chromosome 9 open reading frame 72 with G4C2 nucleotide expansion) and from sporadic cases with no identified mutation, pointing to dysfunction of nBAF chromatin remodelling in multiple forms of ALS.

Published
2017

29. Senescence is a Spi1-induced anti-proliferative mechanism in primary hematopoietic cells

Author

Oliver Bischof, Virginie Penard-Lacronique, Elena Mylonas, Cyril Quiveron, Michela Esposito, Laure Delestré, Christel Guillouf, Hengxiang Cui, Institut Gustave Roussy (IGR), Dynamique moléculaire de la transformation hématopoïétique (Dynamo), Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay, Organisation Nucléaire et Oncogenèse / Nuclear Organization and Oncogenesis, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by the Fondation de France, Inserm, the Institut National du Cancer (INCa-DGOS-INSERM 6043 and PL-BIO-06), ITMO Cancer de l’alliance nationale pour les sciences de la vie et de la santé (AVIESAN), Section régionale de la Ligue Nationale contre le Cancer. L. Delestré was supported by AVIESAN and Fondation de France, H. Cui by Cancéropole Ile-de-France, M. Esposito by the Institut National du Cancer (PL-BIO-06). Quiveron and E. Mylonas by a CDI-Mission (Institut Gustave Roussy)., The authors would like to thank Y. Lecluse, P. Rameau and Z. Maciorowski from the cytometry platform and A. Nicolas, P. Opolon, O. Bawa, S. Arrufat, R. Corre and E. Louvet from the IHC platform. We thank the animal facility housing at Curie and Gustave Roussy Institutes. We thank O. Bernard, M. David for comments on the manuscript and F. Rosselli and F. Moreau-Gachelin for helpful scientific discussions and critical reading of the manuscript., Cheriet Rauline, Samia, and Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, Myeloid, [SDV]Life Sciences [q-bio], Ectopic Gene Expression, MESH: Hematopoietic Stem Cells, Mice, Bone Marrow, hemic and lymphatic diseases, MESH: Animals, Cellular Senescence, Leukemia, Myeloid leukemia, Hematology, Immunohistochemistry, DNA-Binding Proteins, [SDV] Life Sciences [q-bio], Haematopoiesis, medicine.anatomical_structure, MESH: Ectopic Gene Expression, MESH: Bone Marrow, Stem cell, Acute Myeloid Leukemia, Senescence, MESH: Mutation, MESH: Mice, Transgenic, MESH: Trans-Activators, Mice, Transgenic, Biology, Article, Cell Line, 03 medical and health sciences, Proto-Oncogene Proteins, MESH: Cell Proliferation, MESH: Leukemia, medicine, Animals, Humans, MESH: Mice, Cell Proliferation, MESH: Humans, MESH: Immunohistochemistry, MESH: Cellular Senescence, Fibroblasts, Hematopoietic Stem Cells, medicine.disease, MESH: Cell Line, MESH: Proto-Oncogene Proteins, 030104 developmental biology, Cell culture, MESH: Fibroblasts, Mutation, Trans-Activators, Cancer research, MESH: Biomarkers, Bone marrow, Biomarkers, MESH: DNA-Binding Proteins
Abstract

International audience; Transcriptional deregulation caused by epigenetic or genetic alterations is a major cause of leukemic transformation. The Spi1/PU.1 transcription factor is a key regulator of many steps of hematopoiesis, and limits self-renewal of hematopoietic stem cells. The deregulation of its expression or activity contributes to leukemia, in which Spi1 can be either an oncogene or a tumor suppressor. Herein we explored whether cellular senescence, an anti-tumoral pathway that restrains cell proliferation, is a mechanism by which Spi1 limits hematopoietic cell expansion, and thus prevents the development of leukemia. We show that Spi1 overexpression triggers cellular senescence both in primary fibroblasts and hematopoietic cells. Erythroid and myeloid lineages are both prone to Spi1-induced senescence. In hematopoietic cells, Spi1-induced senescence requires its DNA-binding activity and a functional p38MAPK14 pathway but is independent of a DNA-damage response. In contrast, in fibroblasts, Spi1-induced senescence is triggered by a DNA-damage response. Importantly, using our well-established Spi1 transgenic leukemia mouse model, we demonstrate that Spi1 overexpression also induces senescence in erythroid progenitors of the bone marrow in vivo before the onset of the pre-leukemic phase of erythroleukemia. Remarkably, the senescence response is lost during the progression of the disease and erythroid blasts do not display a higher expression of Dec1 and CDKN1A, two of the induced senescence markers in young animals. These results bring indirect evidence that leukemia develops from cells which have bypassed Spi1-induced senescence. Overall, our results reveal senescence as a Spi1-induced anti-proliferative mechanism that may be a safeguard against the development of acute myeloid leukemia.

Published
2017

30. Dynamic stepwise opening of integron attC DNA hairpins by SSB prevents toxicity and ensures functionality

Author

Michael Schlierf, Didier Mazel, Aleksandra Nivina, Bevan L. Cheeseman, Maj Svea Grieb, Andreas Hartmann, Technische Universität Dresden = Dresden University of Technology (TU Dresden), Plasticité du Génome Bactérien - Bacterial Genome Plasticity (PGB), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5), Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG), Max-Planck-Gesellschaft, German Federal Ministry of Education and Research [BMBF 03Z2EN11 to M.S.], Franco-German PROCOPE program of the German Academic exchange service [DAAD 55931827 to M.S.], French Ministry of Foreign Affairs [PHC 28351PC to D.M.], Dresden International Graduate School for Biomedicine and Bioengineering [DFG GS97 to M.S.G. and B.L.C.], Institut Pasteur, Centre National de la Recherche Scientique, Fondation pour la Recherche Médicale [FDT20150532465 to A.N.], Doctoral School 'Frontières du Vivant' funded by the University Paris Descartes. Funding for open access charge: Institutional funds (to M.S.)., We thank Céline Loot and all members of the Schlierf group for discussions, in particular Georg Krainer and Varsha Natarajan for assistance in the experimental design and Philip Gröger and Marko Swoboda for assistance in the data analysis. We thank Bénédicte Michel for her helpful comments on the manuscript. We gratefully acknowledge Iain Patten and the participants of the 'writing for publication' workshop for their support in writing this manuscript., and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
DNA, Bacterial, 0301 basic medicine, MESH: Integrases, Base pair, [SDV]Life Sciences [q-bio], 030106 microbiology, DNA, Single-Stranded, MESH: Escherichia coli Proteins, Genome Integrity, Repair and Replication, Biology, DNA-binding protein, MESH: Attachment Sites, Microbiological, Integrons, 03 medical and health sciences, chemistry.chemical_compound, Genetics, Recombinase, MESH: Protein Binding, Protein secondary structure, Gene, Polymerase, Integrases, Escherichia coli Proteins, DNA replication, MESH: DNA, Bacterial, Cell biology, MESH: Integrons, DNA-Binding Proteins, 030104 developmental biology, MESH: DNA, Single-Stranded, MESH: Nucleic Acid Conformation, chemistry, Attachment Sites, Microbiological, biology.protein, Nucleic Acid Conformation, DNA, MESH: DNA-Binding Proteins, Protein Binding
Abstract

International audience; Biologically functional DNA hairpins are found in archaea, prokaryotes and eukaryotes, playing essential roles in various DNA transactions. However, during DNA replication, hairpin formation can stall the polymerase and is therefore prevented by the single-stranded DNA binding protein (SSB). Here, we address the question how hairpins maintain their functional secondary structure despite SSB's presence. As a model hairpin, we used the recombinogenic form of the attC site, essential for capturing antibiotic-resistance genes in the integrons of bacteria. We found that attC hairpins have a conserved high GC-content near their apical loop that creates a dynamic equilibrium between attC fully opened by SSB and a partially structured attC-6-SSB complex. This complex is recognized by the recombinase IntI, which extrudes the hairpin upon binding while displacing SSB. We anticipate that this intriguing regulation mechanism using a base pair distribution to balance hairpin structure formation and genetic stability is key to the dissemination of antibiotic resistance genes among bacteria and might be conserved among other functional hairpins.

Published
2017

31. Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination

Author

Lenden Hasse, Hélène, Lescale, Chloé, Bianchi, Joy, Yu, Wei, Bedora-Faure, Marie, Deriano, Ludovic, Intégrité du génome, immunité et cancer - Genome integrity, Immunity and Cancer, Institut Pasteur [Paris], Cellule Pasteur, Université Paris Diderot - Paris 7 (UPD7)-PRES Sorbonne Paris Cité, This work was supported by the Institut Pasteur, the European Research Council under the ERC starting grant agreement # 310917 and the Institut National du Cancer (# PLBIO16-181). J.J.B. is a BioSPC/Université Paris 5 doctoral Fellow., We thank Bernardo Reina San Martin, Erika Brunet and Jean-Pierre de Villartay for initial discussion and reagents and the Institut Pasteur Cytometry Platform and Center for Human Immunology for their help., European Project: 310917,EC:FP7:ERC,ERC-2012-StG_20111109,LYMPHOONCOGENOMICS(2013), and Institut Pasteur [Paris] (IP)

Subjects
MESH: Gene Editing, MESH: G1 Phase Cell Cycle Checkpoints, MESH: CRISPR-Cas Systems, DNA End-Joining Repair, CRISPR-Associated Proteins, MESH: DNA Breaks, Double-Stranded, Apoptosis, MESH: Genotype, hemic and lymphatic diseases, MESH: Protein Kinase Inhibitors, Clustered Regularly Interspaced Short Palindromic Repeats, DNA Breaks, Double-Stranded, MESH: Animals, Cell Line, Transformed, Gene Editing, v-Abl transformed pro-B cells, DNA-Binding Proteins, MESH: Recombinational DNA Repair, Phenotype, Proto-Oncogene Proteins c-bcl-2, [SDV.IMM.IA]Life Sciences [q-bio]/Immunology/Adaptive immunology, Imatinib Mesylate, Genotype, MESH: Imatinib Mesylate, Recombination-activating gene (RAG) endonuclease, [SDV.CAN]Life Sciences [q-bio]/Cancer, CRISPR/Cas9-mediated gene knock-out, MESH: Phenotype, Article, MESH: Mice, Inbred C57BL, MESH: Homeodomain Proteins, Animals, MESH: Oncogene Proteins v-abl, MESH: Cell Line, Transformed, Oncogene Proteins v-abl, Protein Kinase Inhibitors, neoplasms, Homeodomain Proteins, Precursor Cells, B-Lymphoid, Nonhomologous end joining (NHEJ), MESH: Apoptosis, MESH: CRISPR-Associated Proteins, Recombinational DNA Repair, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, MESH: DNA End-Joining Repair, G1 Phase Cell Cycle Checkpoints, V(D)J recombination, Mice, Inbred C57BL, MESH: Proto-Oncogene Proteins c-bcl-2, MESH: Precursor Cells, B-Lymphoid, MESH: Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems, MESH: DNA-Binding Proteins
Abstract

International audience; Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair.

Published
2017

32. Bacterial transformation: ComFA is a DNA-dependent ATPase that forms complexes with ComFC and DprA

Author

Diallo, Amy, Foster, Hannah, Gromek, Katarzyna, Perry, Thomas, Dujeancourt, Annick, Krasteva, Petya, Gubellini, Francesca, Falbel, Tanya, Burton, Briana, Fronzes, Rémi, Biologie Structurale de la Sécrétion Bactérienne, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC), University of Wisconsin-Madison, This work was funded by the Centre National de la Recherche Scientifique and the European Research Council (MolStructTransfo, 281149) to RF and by a grant from the Rita Allen Foundation to BMB., European Project: 281149,EC:FP7:ERC,ERC-2011-StG_20101109,MOLSTRUCTTRANSFO(2012), and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
[PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], DNA, Single-Stranded, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Homologous Recombination, Bacterial Proteins, MESH: Adenosine Triphosphatases, [CHIM.CRIS]Chemical Sciences/Cristallography, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Homologous Recombination, MESH: Bacterial Proteins, Adenosine Triphosphatases, Recombination, Genetic, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: DNA, Membrane Proteins, DNA, DNA-Binding Proteins, Rec A Recombinases, Streptococcus pneumoniae, MESH: DNA, Single-Stranded, MESH: Rec A Recombinases, MESH: Recombination, Genetic, Transformation, Bacterial, MESH: Membrane Proteins, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], MESH: Transformation, Bacterial, MESH: Streptococcus pneumoniae, MESH: DNA-Binding Proteins, Protein Binding
Abstract

International audience; Pneumococcal natural transformation contributes to genomic plasticity, antibiotic resistance development and vaccine escape. Streptococcus pneumoniae, like many other naturally transformable species, has evolved sophisticated protein machinery for the binding and uptake of DNA. Two proteins encoded by the comF operon, ComFA and ComFC, are involved in transformation but their exact molecular roles remain unknown. In this study, we provide experimental evidence that ComFA binds to single stranded DNA (ssDNA) and has ssDNA-dependent ATPase activity. We show that both ComFA and ComFC are essential for the transformation process in pneumococci. Moreover, we show that these proteins interact with each other and with other proteins involved in homologous recombination, such as DprA, thus placing the ComFA-ComFC duo at the interface between DNA uptake and DNA recombination during transformation.

Published
2017

33. Structural and functional characterization of the PNKP–XRCC4–LigIV DNA repair complex

Author

David C. Schriemer, Cortt G. Piett, Michael Weinfeld, J. N. Mark Glover, Shujuan Fang, Ruiqiong Ye, John A. Tainer, Ross A. Edwards, Zahra Havali-Shahriari, R. Daniel Aceytuno, Fatima Javed, Rajam S. Mani, Martial Rey, Michal Hammel, Susan P. Lees-Miller, Department of Biochemistry [Edmonton, AB, Canada] (Faculty of Medicine and Dentistry), University of Alberta, Department of Biochemistry and Molecular Biology, Southern Alberta Cancer Research Institute, University of Calgary, Cross Cancer Institute and Department of Oncology, Molecular Biophysics and Integrated Bioimaging Division [Berkeley, CA, USA], and Lawrence Berkeley National Laboratory [Berkeley] (LBNL)

Subjects
Models, Molecular, 0301 basic medicine, DNA End-Joining Repair, MESH: Deuterium, Protein Conformation, Developmental Disabilities, MESH: Catalytic Domain, medicine.disease_cause, Mass Spectrometry, MESH: Recombinant Proteins, DNA Ligase ATP, chemistry.chemical_compound, Protein structure, MESH: Protein Conformation, X-Ray Diffraction, Structural Biology, Catalytic Domain, Protein Interaction Mapping, MESH: Syndrome, Phosphorylation, chemistry.chemical_classification, Mutation, MESH: X-Ray Diffraction, Syndrome, DNA repair protein XRCC4, MESH: Seizures, Recombinant Proteins, Cell biology, DNA-Binding Proteins, Phosphotransferases (Alcohol Group Acceptor), [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: DNA Repair Enzymes, Biochemistry, Microcephaly, MESH: Models, Molecular, Protein Binding, DNA damage, Mutation, Missense, Biology, MESH: Microcephaly, 03 medical and health sciences, MESH: DNA Ligase ATP, Seizures, DNA repair complex, Scattering, Small Angle, Genetics, medicine, Humans, Point Mutation, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Scattering, Small Angle, MESH: Point Mutation, MESH: DNA Damage, MESH: Mass Spectrometry, DNA ligase, MESH: Mutation, Missense, MESH: Humans, 030102 biochemistry & molecular biology, MESH: Phosphorylation, Point mutation, MESH: Protein Interaction Mapping, MESH: DNA End-Joining Repair, MESH: Multiprotein Complexes, Deuterium, MESH: Phosphotransferases (Alcohol Group Acceptor), DNA Repair Enzymes, 030104 developmental biology, chemistry, MESH: Developmental Disabilities, Multiprotein Complexes, MESH: Protein Processing, Post-Translational, Protein Processing, Post-Translational, DNA, MESH: DNA-Binding Proteins, DNA Damage
Abstract

International audience; Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4-LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP-XRCC4-LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4-LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4-LigIV that regulate PNKP recruitment and activity within NHEJ.

Published
2017

34. Translation of Expanded CGG Repeats into FMRpolyG Is Pathogenic and May Contribute to Fragile X Tremor Ataxia Syndrome

Author

Marie-Christine Birling, Chantal Sellier, Frank Ruffenach, Cécile Martinat, Marie Wattenhofer-Donzé, Stéphane Viville, Ronald A.M. Buijsen, Mustapha Oulad-Abdelghani, Hamid Meziane, Peter K. Todd, Alexandre Vincent, Guillaume Pavlovic, Pascal Eberling, Hugues Jacobs, Verónica Martínez-Cerdeño, Flora Tassone, Marie-France Champy, Laura Jung, Fang He, Nicolas Charlet-Berguerand, Philippe Tropel, Mathieu Anheim, Angeline Gaucherot, Tania Sorg, Renate K. Hukema, Mathilde Joint, Rob Willemsen, Sam Natla, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Erasmus University Medical Center [Rotterdam] (Erasmus MC), University of Michigan [Ann Arbor], University of Michigan System, Institut Clinique de la Souris (ICS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Strasbourg (UNISTRA), Hôpital de Hautepierre [Strasbourg], University of California [Davis] (UC Davis), University of California, Laboratoire de Diagnostic Génétique [CHU Strasbourg], Université de Strasbourg (UNISTRA)-CHU Strasbourg, Nouvel Hôpital Civil de Strasbourg, Institut de Parasitologie et de Pathologie Tropicale (IPPTS), Université de Strasbourg (UNISTRA), Université d'Évry-Val-d'Essonne (UEVE), Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Saclay-Généthon, Association française contre les myopathies (AFM-Téléthon), Centre for Integrative Biology - CBI (Inserm U964 - CNRS UMR7104 - IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), French National Infrastructure for Mouse Phenogenomics (PHENOMIN), CHU Strasbourg, University of California (UC), Dieterle, Stéphane, and Clinical Genetics

Subjects
Male, 0301 basic medicine, Untranslated region, MESH: Ataxia, [SDV]Life Sciences [q-bio], MESH: Tremor, MESH: Nuclear Lamina, Pathogenesis, Fragile X Mental Retardation Protein, Mice, [SCCO]Cognitive science, Tremor, MESH: Trinucleotide Repeat Expansion, MESH: Animals, Induced pluripotent stem cell, ComputingMilieux_MISCELLANEOUS, Genetics, Fragile X Tremor/Ataxia Syndrome, MESH: Peptides, MESH: Real-Time Polymerase Chain Reaction, General Neuroscience, neurodegeneration, Brain, Translation (biology), 3. Good health, DNA-Binding Proteins, [SDV] Life Sciences [q-bio], MESH: Protein Biosynthesis, Nuclear lamina, MESH: Membrane Proteins, MESH: Fragile X Syndrome, medicine.symptom, congenital, hereditary, and neonatal diseases and abnormalities, near-cognate codon, Ataxia, MESH: Mice, Transgenic, Neuroscience(all), Mice, Transgenic, Biology, Real-Time Polymerase Chain Reaction, MESH: Fragile X Mental Retardation Protein, Article, MESH: Brain, 03 medical and health sciences, RAN translation, medicine, Animals, Humans, RNA, Messenger, MESH: Mice, MESH: RNA, Messenger, Nuclear Lamina, MESH: Humans, Membrane Proteins, RNA, [SCCO] Cognitive science, microsatellite expansion, MESH: Male, nervous system diseases, 030104 developmental biology, Fragile X Syndrome, Protein Biosynthesis, Peptides, Trinucleotide Repeat Expansion, MESH: DNA-Binding Proteins
Abstract

Summary Fragile X-associated tremor/ataxia syndrome (FXTAS) is a neurodegenerative disorder caused by a limited expansion of CGG repeats in the 5′ UTR of FMR1. Two mechanisms are proposed to cause FXTAS: RNA gain-of-function, where CGG RNA sequesters specific proteins, and translation of CGG repeats into a polyglycine-containing protein, FMRpolyG. Here we developed transgenic mice expressing CGG repeat RNA with or without FMRpolyG. Expression of FMRpolyG is pathogenic, while the sole expression of CGG RNA is not. FMRpolyG interacts with the nuclear lamina protein LAP2β and disorganizes the nuclear lamina architecture in neurons differentiated from FXTAS iPS cells. Finally, expression of LAP2β rescues neuronal death induced by FMRpolyG. Overall, these results suggest that translation of expanded CGG repeats into FMRpolyG alters nuclear lamina architecture and drives pathogenesis in FXTAS., Highlights • CGG repeats in the 5′ UTR of FMR1 are translated through initiation to an ACG codon • Translation of CGG repeats in the polyglycine protein, FMRpolyG, is toxic in mice • FMRpolyG binds and disrupts protein of the nuclear lamina, Sellier et al. show that translation of expanded CGG repeats located in the 5′ UTR of the FMR1 gene require an upstream ACG near-cognate initiation codon. Translation of CGG repeats into a short polyglycine-containing protein, FMRpolyG, is pathogenic in mouse models.

Published
2017

35. The Integron Integrase Efficiently Prevents the Melting Effect of Escherichia coli Single-Stranded DNA-Binding Protein on Folded attC Sites

Author

Céline Loot, David Bikard, Vincent Parissi, Jihane Amarir-Bouhram, Jose Antonio Escudero, Didier Mazel, Plasticité du Génome Bactérien - Bacterial Genome Plasticity (PGB), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Microbiologie cellulaire et moléculaire et pathogénicité (MCMP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), This work was supported by the Institut Pasteur, the Centre National de la Recherche Scientifique (CNRS-UMR3525), the European Union Seventh Framework Programme (FP7-HEALTH-2011-single-stage), and the Evolution and Transfer of Antibiotic Resistance (EvoTAR), European Project: 282004,EC:FP7:HEALTH,FP7-HEALTH-2011-single-stage,EVOTAR(2011), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Microbiologie Fondamentale et Pathogénicité (MFP)

Subjects
DNA, Bacterial, MESH: Integrases, Plasma protein binding, medicine.disease_cause, Integron, MESH: Attachment Sites, Microbiological, Microbiology, DNA-binding protein, Integrons, 03 medical and health sciences, chemistry.chemical_compound, Escherichia coli, medicine, MESH: Protein Binding, Molecular Biology, Gene, 030304 developmental biology, Recombination, Genetic, Genetics, 0303 health sciences, Integrases, biology, MESH: Escherichia coli, 030306 microbiology, Escherichia coli Proteins, Articles, MESH: DNA, Bacterial, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, MESH: Integrons, Integrase, DNA-Binding Proteins, MESH: Nucleic Acid Conformation, chemistry, Attachment Sites, Microbiological, biology.protein, Nucleic Acid Conformation, MESH: Recombination, Genetic, MESH: DNA-Binding Proteins, DNA, Protein Binding
Abstract

Integrons play a major role in the dissemination of antibiotic resistance genes among bacteria. Rearrangement of gene cassettes occurs by recombination between attI and attC sites, catalyzed by the integron integrase. Integron recombination uses an unconventional mechanism involving a folded single-stranded attC site. This site could be a target for several host factors and more precisely for proteins able to bind single-stranded DNA. One of these, Escherichia coli single-stranded DNA-binding protein (SSB), regulates many DNA processes. We studied the influence of this protein on integron recombination. Our results show the ability of SSB to strongly bind folded attC sites and to destabilize them. This effect was observed only in the absence of the integrase. Indeed, we provided evidence that the integrase is able to counterbalance the observed effect of SSB on attC site folding. We showed that IntI1 possesses an intrinsic property to capture attC sites at the moment of their extrusion, stabilizing them and recombining them efficiently. The stability of DNA secondary structures in the chromosome must be restrained to avoid genetic instability (mutations or deletions) and/or toxicity (replication arrest). SSB, which hampers attC site folding in the absence of the integrase, likely plays an important role in maintaining the integrity and thus the recombinogenic functionality of the integron attC sites. We also tested the RecA host factor and excluded any role of this protein in integron recombination.

Published
2014

36. Gata3 drives development of ROR gamma t(+) group 3 innate lymphoid cells

Author

Roel G. J. Klein Wolterink, Wei Xu, Naoko Satoh-Takayama, Rudi W. Hendriks, Christian A. J. Vosshenrich, James P. Di Santo, Nicolas Serafini, Immunité Innée - Innate Immunity, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Department of Pulmonary Medicine, Erasmus University Medical Center [Rotterdam] (Erasmus MC), R.G.J. Klein Wolterink is supported by a scholarship from the Ligue Nationale Contre le Cancer (LNCC, France). This work is supported by grants from the Institut Pasteur, Institut National de la Santé et de la Recherche Médicale, LNCC (Equipe Labellisée Ligue Contre le Cancer), and Agence National pour la Recherche (Program 'Blanc' Gut_ILC)., Pulmonary Medicine, Di Santo, James, and Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
MESH: Nuclear Receptor Subfamily 1, Group F, Member 3, MESH: Citrobacter rodentium, Adaptive Immunity, MESH: Mice, Knockout, Fetal Development, Mice, 0302 clinical medicine, MESH: Pregnancy, RAR-related orphan receptor gamma, Pregnancy, Immunology and Allergy, MESH: Animals, MESH: Enterobacteriaceae Infections, Mice, Knockout, 0303 health sciences, Innate lymphoid cell, GATA3, Enterobacteriaceae Infections, Nuclear Receptor Subfamily 1, Group F, Member 3, Acquired immune system, DNA-Binding Proteins, Haematopoiesis, medicine.anatomical_structure, Lymphatic system, Liver, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Immunity, Innate, Female, Stem cell, MESH: Fetal Development, MESH: Interleukins, [SDV.IMM] Life Sciences [q-bio]/Immunology, MESH: Mice, Transgenic, T cell, Immunology, MESH: Lymphocyte Subsets, Mice, Transgenic, GATA3 Transcription Factor, Biology, 03 medical and health sciences, MESH: Mice, Inbred C57BL, MESH: GATA3 Transcription Factor, medicine, Animals, MESH: Mice, Immunity, Mucosal, 030304 developmental biology, Interleukins, Brief Definitive Report, Immunity, Innate, Lymphocyte Subsets, Mice, Inbred C57BL, MESH: Immunity, Mucosal, Citrobacter rodentium, MESH: Female, MESH: Adaptive Immunity, MESH: DNA-Binding Proteins, 030215 immunology, MESH: Liver
Abstract

The transcription factor Gata3 is required for the generation of group 3 innate lymphoid cells (ILC3) that protect mucosal surfaces., Group 3 innate lymphoid cells (ILC3) include IL-22–producing NKp46+ cells and IL-17A/IL-22–producing CD4+ lymphoid tissue inducerlike cells that express RORγt and are implicated in protective immunity at mucosal surfaces. Whereas the transcription factor Gata3 is essential for T cell and ILC2 development from hematopoietic stem cells (HSCs) and for IL-5 and IL-13 production by T cells and ILC2, the role for Gata3 in the generation or function of other ILC subsets is not known. We found that abundant GATA-3 protein is expressed in mucosa-associated ILC3 subsets with levels intermediate between mature B cells and ILC2. Chimeric mice generated with Gata3-deficient fetal liver hematopoietic precursors lack all intestinal RORγt+ ILC3 subsets, and these mice show defective production of IL-22 early after infection with the intestinal pathogen Citrobacter rodentium, leading to impaired survival. Further analyses demonstrated that ILC3 development requires cell-intrinsic Gata3 expression in fetal liver hematopoietic precursors. Our results demonstrate that Gata3 plays a generalized role in ILC lineage determination and is critical for the development of gut RORγt+ ILC3 subsets that maintain mucosal barrier homeostasis. These results further extend the paradigm of Gata3-dependent regulation of diversified innate ILC and adaptive T cell subsets.

Published
2014

37. An evolutionary conserved interaction between the Gcm transcription factor and the SF1 nuclear receptor in the female reproductive system

Author

Angela Giangrande, Wael Bazzi, Claude Delaporte, Pierre B. Cattenoz, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), and Cattenoz, Pierre

Subjects
0301 basic medicine, Receptors, Steroid, Exocrine gland, Regulator, Female reproductive system, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Mice, 0302 clinical medicine, Drosophila Proteins, MESH: Animals, Reproductive system, ComputingMilieux_MISCELLANEOUS, Genetics, 030219 obstetrics & reproductive medicine, Multidisciplinary, MESH: Infertility, Female, [SDV.BDD.EO] Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis, Cell Differentiation, Genitalia, Female, MESH: Transcription Factors, Biological Evolution, DNA-Binding Proteins, Drosophila melanogaster, medicine.anatomical_structure, Female, RNA Splicing Factors, Infertility, Female, Protein Binding, Infertility, MESH: Cell Differentiation, endocrine system, MESH: Drosophila Proteins, MESH: Biological Evolution, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Article, MESH: Drosophila melanogaster, 03 medical and health sciences, Spermatheca, medicine, Animals, MESH: Protein Binding, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Transcription factor, MESH: Mice, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, medicine.disease, MESH: RNA Splicing Factors, 030104 developmental biology, [SDV.BDD.EO]Life Sciences [q-bio]/Development Biology/Embryology and Organogenesis, Nuclear receptor, MESH: Genitalia, Female, MESH: Female, MESH: DNA-Binding Proteins, Transcription Factors, MESH: Receptors, Steroid
Abstract

NR5A1 is essential for the development and for the function of steroid producing glands of the reproductive system. Moreover, its misregulation is associated with endometriosis, which is the first cause of infertility in women. Hr39, the Drosophila ortholog of NR5A1, is expressed and required in the secretory cells of the spermatheca, the female exocrine gland that ensures fertility by secreting substances that attract and capacitate the spermatozoids. We here identify a direct regulator of Hr39 in the spermatheca: the Gcm transcription factor. Furthermore, lack of Gcm prevents the production of the secretory cells and leads to female sterility in Drosophila. Hr39 regulation by Gcm seems conserved in mammals and involves the modification of the DNA methylation profile of mNr5a1. This study identifies a new molecular pathway in female reproductive system development and suggests a role for hGCM in the progression of reproductive tract diseases in humans.

Published
2016

38. Single molecule tracking of Ace1p in Saccharomyces cerevisiae defines a characteristic residence time for non-specific interactions of transcription factors with chromatin

Author

Tatiana S. Karpova, James G. McNally, David A. Ball, Tatsuya Morisaki, Gunjan D. Mehta, Davide Mazza, Florian Mueller, Ronit Salomon-Kent, National Cancer Institute [Bethesda] (NCI-NIH), National Institutes of Health [Bethesda] (NIH), San Raffaele Scientific Institute, Vita-Salute San Raffaele University and Center for Translational Genomics and Bioinformatics, Colorado State University [Fort Collins] (CSU), Imagerie et Modélisation, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institute for Soft Matter and Functional Materials [Berlin], Helmholtz-Zentrum Berlin für Materialien und Energie GmbH (HZB), Intramural Research Program of National Institutes of Health (NIH), National Cancer Institute (NCI) and Center for Cancer Research (CCR). Funding for open access charge: Intramural Research Program of National Institutes of Health (NIH)., The authors would like to thank Dr Luke D. Lavis and Dr Jonatan B. Grimm (Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA) for JF HaloTag ligands. The authors acknowledge Euroscarf for providing pUG6 plasmid., Ball, Da, Mehta, Gd, Salomon-Kent, R, Mazza, D, Morisaki, T, Mueller, F, Mcnally, Jg, Karpova, Ts, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Time Factors, [SDV]Life Sciences [q-bio], dna, MESH: Heat-Shock Proteins, Genome, Histones, chemistry.chemical_compound, MESH: Saccharomyces cerevisiae Proteins, Heat-Shock Proteins, transcription factor, Genetics, MESH: Histones, MESH: Transcription Factors, MESH: Saccharomyces cerevisiae, Molecular Imaging, Chromatin, DNA-Binding Proteins, Methods Online, Inhouse research on structure dynamics and function of matter, Saccharomyces cerevisiae Proteins, Fluorophore, Recombinant Fusion Proteins, Saccharomyces cerevisiae, yeasts, Computational biology, Biology, MESH: Chromatin, 03 medical and health sciences, MESH: Recombinant Fusion Proteins, Transcription factor, molecule, MESH: Molecular Imaging, MESH: Metallothionein, MESH: Time Factors, biology.organism_classification, Yeast, 030104 developmental biology, chemistry, chromatin, Metallothionein, blinking, DNA, Function (biology), MESH: DNA-Binding Proteins, Transcription Factors
Abstract

In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12–0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly different DNA-bound proteins and thus may have a common underlying mechanism. We develop new and robust procedure for evaluation of adverse effects of labeling, and new quantitative analysis procedures that significantly improve residence time measurements by accounting for fluorophore blinking. Our results provide a framework for the reliable performance and analysis of single molecule TF experiments in yeast.

Published
2016

39. Peroxisome proliferator-activated receptor alpha deficiency impairs regulatory T cell functions: Possible application in the inhibition of melanoma tumor growth in mice

Author

François Ghiringhelli, Narcisse Zwetyenga, Naim Akhtar Khan, Kabirou Moutairou, Françoise Salvadori, Aziz Hichami, Akadiri Yessoufou, Lipides - Nutrition - Cancer (U866) ( LNC ), Université de Bourgogne ( UB ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon ( ENSBANA ), Laboratoire de Biochimie et de Biologie Moléculaire (Université d'Abomey Calavi, Cotonou, Bénin) ( LBBM ), Université d'Abomey Calavi, French Ministry of Higher Education and Research, National Cancer Institute (1-23651-07), Lipides - Nutrition - Cancer (U866) (LNC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Bourgogne (UB)-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, Laboratoire de Biochimie et de Biologie Moléculaire (Université d'Abomey Calavi, Cotonou, Bénin) (LBBM), University of Abomey Calavi (UAC), and Université de Bourgogne (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Ecole Nationale Supérieure de Biologie Appliquée à la Nutrition et à l'Alimentation de Dijon (ENSBANA)

Subjects
0301 basic medicine, Male, Adoptive cell transfer, MESH: Tumor Burden, B16 melanoma tumor, Melanoma, Experimental, MESH: T-Lymphocyte Subsets, CD4(+)CD25(+) regulatory T cells, Biochemistry, MESH: Mice, Knockout, Immunotherapy, Adoptive, T-Lymphocytes, Regulatory, PPARα, MESH : T-Lymphocytes, Regulatory, Cell Movement, T-Lymphocyte Subsets, MESH: Reverse Transcriptase Polymerase Chain Reaction, MESH : Cell Proliferation, MESH : Cell Movement, MESH: Animals, IL-2 receptor, MESH: PPAR alpha, MESH: Cell Movement, Cells, Cultured, Mice, Knockout, MESH : Melanoma, Experimental, biology, MESH : Tumor Burden, Reverse Transcriptase Polymerase Chain Reaction, MESH : Reverse Transcriptase Polymerase Chain Reaction, FOXP3, hemic and immune systems, General Medicine, MESH: Gene Expression Regulation, Neoplastic, 3. Good health, Tumor Burden, MESH: Melanoma, Experimental, DNA-Binding Proteins, Gene Expression Regulation, Neoplastic, medicine.anatomical_structure, MESH: Immunotherapy, Adoptive, Receptors, Chemokine, MESH : DNA-Binding Proteins, MESH: Cells, Cultured, medicine.medical_specialty, MESH : Receptors, Chemokine, MESH: Cell Line, Tumor, Regulatory T cell, MESH : Gene Expression Regulation, Neoplastic, T cell, MESH : Male, MESH : PPAR alpha, chemical and pharmacologic phenomena, MESH : Mice, Inbred C57BL, MESH : Clonal Anergy, 03 medical and health sciences, MESH: Mice, Inbred C57BL, Internal medicine, MESH: Cell Proliferation, Cell Line, Tumor, MESH : Cells, Cultured, medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, PPAR alpha, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cell Proliferation, Clonal Anergy, Perforin, MESH : Cell Line, Tumor, MESH: T-Lymphocytes, Regulatory, Molecular biology, MESH: Male, MESH : T-Lymphocyte Subsets, Granzyme B, Mice, Inbred C57BL, 030104 developmental biology, Endocrinology, MESH: Clonal Anergy, biology.protein, MESH : Mice, Knockout, MESH : Animals, MESH: Receptors, Chemokine, CD8, MESH: DNA-Binding Proteins, MESH : Immunotherapy, Adoptive
Abstract

International audience; Regulatory T (Treg) cells are important to induce and maintain immunological self-tolerance. Although the progress accomplished in understanding the functional mechanism of Treg cells, intracellular molecules that control the mechanisms of their suppressive capacity are still on investigation. The present study showed that peroxisome proliferator-activated receptor-alpha deficiency impaired the suppressive activity of Treg cells on CD4(+)CD25(-) and CD8(+) T cell proliferation. In Treg cells, PPARα gene deletion also induced a decrease of migratory abilities, and downregulated the expression of chemokine receptors (CCR-4, CCR-8 and CXCR-4) and p27(KIP1) mRNA. Treg cells from PPARα(-/-) mice also lost their anergic property. Since low Treg activity, as observed in PPARα(-/-) mice, is known to be associated with the inhibition of tumor growth, we inoculated these mice with B16 melanoma cells and assessed tumor proliferation. In PPARα(-/-) mice, cancer growth was significantly curtailed, and it was correlated with high expression of granzyme B and perforin mRNA in tumor bed. Degranulation of cytolytic molecules by CD8(+) T cells, assessed by a perforin-release marker CD107a expression, was higher in PPARα(-/-) mice than that in wild-type mice. Tumor-infiltrating lymphocytes (TIL) in melanoma tumors in PPARα(-/-) mice exhibited high pro-inflammatory Th1 phenotype. Consistently, adoptive transfer into lymphopenic RAG2(-/-) mice of total PPARα(-/-)splenic T cells inhibited more the growth rate of B16 tumor than the wild type splenic T cells. Our findings suggest that PPARα deficiency, by diminishing Treg cell functions and upregulating pro-inflammatory T cell phenotype, exerts an in vivo anti-cancer properties.

Published
2016

40. Bioinformatic analysis of the protein/DNA interface

Author

Daniel Svozil, Jiří Černý, Bohdan Schneider, Alexandre G. de Brevern, Agnel Praveen Joseph, Jean-Christophe Gelly, de Brevern, Alexandre G., Institute of Biotechnology AS CR, Laboratory of Informatics and Chemistry, Institute of Chemical Technology [Prague] (ICT), GR-Ex, Laboratoire d'Excellence, GR-Ex, Dynamique des Structures et Interactions des Macromolécules Biologiques (DSIMB), Biologie Intégrée du Globule Rouge (BIGR (UMR_S_1134 / U1134)), Institut National de la Transfusion Sanguine [Paris] (INTS)-Université Paris Diderot - Paris 7 (UPD7)-Université de La Réunion (UR)-Université des Antilles (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de la Transfusion Sanguine [Paris] (INTS)-Université Paris Diderot - Paris 7 (UPD7)-Université de La Réunion (UR)-Université des Antilles (UA)-Institut National de la Santé et de la Recherche Médicale (INSERM), The Czech-France collaboration Barrande [MEB021032], BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF, [P305/ 80 12/1801] from the Czech Science Foundation, and institutional [AV0Z50520701], supported by [MSM 6046137302 to D.S. and P.Č.], and supported by the Ministry of Research (France), University Paris Diderot, Sorbonne Paris Cité (France), National Institute of Blood Transfusion (INTS, France), National Institute of Health and Medical Research (INSERM, France) and 'Investissements d'avenir', Laboratories of Excellence GR-Ex (France) (to J.C.G. and A.G.dB.). Funding for open access charge: Czech Science Foundation and Academy of Sciences of the Czech Republic.

Subjects
Models, Molecular, Protein Conformation, DNA interface, Crystal structure, Plasma protein binding, Pentapeptide repeat, chemistry.chemical_compound, 0302 clinical medicine, structural alphabet: Protein Blocks, MESH: Protein Conformation, Protein structure, Structural Biology, Structural motif, Conformational isomerism, [INFO.INFO-BI] Computer Science [cs]/Bioinformatics [q-bio.QM], Genetics, 0303 health sciences, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], MESH: DNA, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], DNA-Binding Proteins, MESH: Nucleic Acid Conformation, Naked DNA, Data Interpretation, Statistical, MESH: Phosphates, MESH: Models, Molecular, Protein Binding, MESH: Computational Biology, Stereochemistry, Biophysics, Biology, DNA-binding protein, Phosphates, 03 medical and health sciences, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Water, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Transcription factor, 030304 developmental biology, Computational Biology, Water, DNA structure, DNA, Base (topology), Crystallography, chemistry, protein structures, Nucleic acid, Nucleic Acid Conformation, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], DNA interactions, protein, MESH: Data Interpretation, Statistical, 030217 neurology & neurosurgery, MESH: DNA-Binding Proteins
Abstract

Despite extensive studies of the geometry of protein/DNA interfaces the understanding of the affinity and specificity of the protein/DNA interactions remains elusive. We present a novel approach to geometric analysis of protein/nucleic acid interfaces that is based on classification of their local conformations. Protein structures are divided into a series of pentapeptide fragments and each is assigned one of 16 conformers or ‘protein blocks’ [de Brevern et al. Proteins 41, 271 (2000)]. Similarly, each DNA step (unit [base]sugar-phosphate-sugar[base]) is assigned one of ∼20 DNA conformers [Svozil et al. NAR 36, 3690 (2008)]. Significantly, local structures classified into distinct conformers can be represented by symbols so that they can be analyzed more easily than complicated 3D objects. Size of the fragments used for the classification allows analysis of structural features of the interface at a scale intermediate between too detailed interatomic contacts on one side and too crude protein motifs (helix-turn-helix, Zn-finger, ...) on the other. We will present correlations between protein and DNA conformers at their interface from more than 15 hundred protein/DNA crystal structures broken by various criteria of the analyzed structures as protein functional classification, e.g. in GO or Pfam or by crystallographic properties, resolution or overall structure quality.Acknowledgments. This work was supported by Czech-France collaboration Barrande (MEB021032) and Czech Science Foundation (P305/10/2184).

Published
2013

41. Functional insights into the core-TFIIH from a comparative survey

Author

Odile Lecompte, Florence Bedez, Julie D. Thompson, Raymond Ripp, Benjamin Linard, Olivier Poch, Xavier Brochet, Dino Moras, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), and Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Transcription, Genetic, Sequence analysis, Biology, Splicing, [SDV.BID.SPT]Life Sciences [q-bio]/Biodiversity/Systematics, Phylogenetics and taxonomy, Phylogenetic distribution, CDK-activating kinase, Evolution, Molecular, 03 medical and health sciences, Transcription (biology), [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Genetics, Animals, Humans, MESH: Animals, Comparative genomic analysis, MESH: Phylogeny, MESH: Evolution, Molecular, Phylogeny, 030304 developmental biology, 0303 health sciences, MESH: Humans, TFIIH, MESH: Transcription Factor TFIIH, MESH: Transcription, Genetic, 030302 biochemistry & molecular biology, MESH: Multiprotein Complexes, P34, MESH: Protein Subunits, Cyclin-Dependent Kinases, DNA-Binding Proteins, Protein Subunits, MESH: Cyclin-Dependent Kinases, Multiprotein Complexes, RNA splicing, Proteome, P34 2, Transcription factor II H, [SDE.BE]Environmental Sciences/Biodiversity and Ecology, [INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM], Transcription, Transcription Factor TFIIH, Repair, MESH: DNA-Binding Proteins
Abstract

International audience; TFIIH is a eukaryotic complex composed of two subcomplexes, the CAK (Cdk activating kinase) and the core-TFIIH. The core-TFIIH, composed of seven subunits (XPB, XPD, P62, P52, P44, P34, and P8), plays a crucial role in transcription and repair. Here, we performed an extended sequence analysis to establish the accurate phy-logenetic distribution of the core-TFIIH in 63 eukaryotic organisms. In spite of the high conservation of the seven subunits at the sequence and genomic levels, the non-enzymatic P8, P34, P52 and P62 are absent from one or a few unicellular species. To gain insight into their respective roles, we undertook a comparative genomic analysis of the whole proteome to identify the gene sets sharing similar presence/absence patterns. While little information was inferred for P8 and P62, our studies confirm the known role of P52 in repair and suggest for the first time the implication of the core TFIIH in mRNA splicing via P34.

Published
2013
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42. Higher-Order Looping and Nuclear Organization of Tcra Facilitate Targeted RAG Cleavage and Regulated Rearrangement in Recombination Centers

Author

David G. Schatz, Iannis Aifantis, Panagiotis Ntziachristos, Mariann Micsinai, Joy M. H. Wang, Ludovic Deriano, Yuval Kluger, Jeffrey A. Jeddeloh, Elphège P. Nora, Matthew Rodesch, Julie Chaumeil, Yanhong Ji, Jane A. Skok, New York University School of Medicine (NYU), New York University School of Medicine, NYU System (NYU)-NYU System (NYU), Développement lymphocytaire et Oncogénèse, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Génétique et Biologie du Développement, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Roche NimbleGen Inc., Yale Cancer Center, Yale School of Medicine [New Haven, Connecticut] (YSM), Yale University [New Haven], Howard Hughes Medical Institute (HHMI), This work is supported by the National Institute ofHealth: R01GM086852 (to J.A.S.), R37AI32524 (to D.G.S.), RO1CA133379,RO1CA105129, RO1CA149655, and RO1GM088847 (to I.A.). J.A.S. is aLeukemia & Lymphoma Society (LLS) scholar. J.C. is an Irvington Institute Fellow of the Cancer Research Institute. M.M. was supported by an NSFIGERT 0333389. L.D. was an LLS Fellow. E.P.N. was supported by the FrenchMinistry of Research and the Association pour la Recherche sur le Cancer(France). I.A. is also supported by the Leukemia & Lymphoma Society (TRPgrant), The V Foundation for Cancer Research, and the Dana Foundation.D.G.S. is an Investigator, and I.A. is an Early Career Scientist of the HowardHughes Medical Institute. The authors from Roche NimbleGen, Inc. (M.J.R.and J.A.J.) recognize a competing interest in this publication as employeesof the company., Lassailly-Bondaz, Anne, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), and Yale University School of Medicine

Subjects
Genome instability, MESH: V(D)J Recombination, Ataxia Telangiectasia Mutated Proteins, Inbred C57BL, MESH: Mice, Knockout, Tumor Suppressor Proteins/metabolism, Mice, 0302 clinical medicine, Transcription (biology), Receptors, Recombinase, Medicine and Health Sciences, MESH: Animals, MESH: Ataxia Telangiectasia Mutated Proteins, lcsh:QH301-705.5, DNA-Binding Proteins/genetics/*metabolism, Genetics, MESH: Histones, 0303 health sciences, MESH: Mice, Inbred CBA, MESH: Receptors, Antigen, T-Cell, alpha-beta, biology, MESH: Genomic Instability, V(D)J recombination, Protein-Serine-Threonine Kinases/metabolism, Chromatin, alpha-beta/genetics/*metabolism, Histone, Antigen, [SDV.IMM]Life Sciences [q-bio]/Immunology, MESH: Cell Nucleus, [SDV.IMM] Life Sciences [q-bio]/Immunology, Knockout, Cleavage (embryo), DNA-binding protein, MESH: Genetic Loci, General Biochemistry, Genetics and Molecular Biology, Genomic Instability, MESH: Protein-Serine-Threonine Kinases, 03 medical and health sciences, MESH: Cell Cycle Proteins, MESH: Mice, Inbred C57BL, MESH: Homeodomain Proteins, Homeodomain Proteins/genetics/*metabolism, Animals, MESH: Tumor Suppressor Proteins, MESH: Mice, Alleles, 030304 developmental biology, MESH: DNA Damage, Histones/genetics, Cell Cycle Proteins/metabolism, MESH: Alleles, Inbred CBA, Biology and Life Sciences, T-Cell, lcsh:Biology (General), Genetic Loci, Cell Nucleus/metabolism, biology.protein, 030217 neurology & neurosurgery, MESH: DNA-Binding Proteins, V(D)J Recombination, DNA Damage
Abstract

International audience; V(D)J recombination is essential for generating a diverse array of B and T cell receptors that can recognize and combat foreign antigens. As with any recombination event, tight control is essential to prevent the occurrence of genetic anomalies that drive cellular transformation. One important aspect of regulation is directed targeting of the RAG recombinase. Indeed, RAG accumulates at the 3' end of individual antigen receptor loci poised for rearrangement; however, it is not known whether focal binding is involved in regulating cleavage, and what mechanisms lead to enrichment of RAG in this region. Here, we show that monoallelic looping out of the 3' end of the T cell receptor α (Tcra) locus, coupled with transcription and increased chromatin/nuclear accessibility, is linked to focal RAG binding and ATM-mediated regulation of monoallelic cleavage on looped-out 3' regions. Our data identify higher-order loop formation as a key determinant of directed RAG targeting and the maintenance of genome stability.

Published
2013
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43. The Glide/Gcm fate determinant controls initiation of collective cell migration by regulating Frazzled

Author

Tripti Gupta, Angela Giangrande, Arun Kumar, K. VijayRaghavan, Pierre B. Cattenoz, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Tata Institute for Fundamental Research (TIFR), and Cattenoz, Pierre

Subjects
0301 basic medicine, MESH: Drosophila, Organogenesis, Cell, MESH: Wings, Animal, Cell Movement, Drosophila Proteins, Wings, Animal, MESH: Animals, Biology (General), Receptor, MESH: Cell Movement, MESH: Receptors, Cell Surface, collective cell migration, D. melanogaster, General Neuroscience, GCM transcription factors, General Medicine, Anatomy, MESH: Transcription Factors, [SDV.BDD.MOR] Life Sciences [q-bio]/Development Biology/Morphogenesis, Cell biology, DNA-Binding Proteins, medicine.anatomical_structure, Medicine, Drosophila, MESH: Neuroglia, frazzled, Stem cell, Netrin Receptors, Neuroglia, Research Article, animal structures, QH301-705.5, MESH: Drosophila Proteins, Science, Receptors, Cell Surface, glide/gcm, Biology, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, developmental biology, stem cells, medicine, Animals, Transcription factor, General Immunology and Microbiology, Collective cell migration, glial cell, Chemotaxis, [SDV.BDD.MOR]Life Sciences [q-bio]/Development Biology/Morphogenesis, MESH: Netrin Receptors, MESH: Organogenesis, Developmental Biology and Stem Cells, 030104 developmental biology, nervous system, Developmental biology, MESH: DNA-Binding Proteins, Transcription Factors
Abstract

Collective migration is a complex process that contributes to build precise tissue and organ architecture. Several molecules implicated in cell interactions also control collective migration, but their precise role and the finely tuned expression that orchestrates this complex developmental process are poorly understood. Here, we show that the timely and threshold expression of the Netrin receptor Frazzled triggers the initiation of glia migration in the developing Drosophila wing. Frazzled expression is induced by the transcription factor Glide/Gcm in a dose-dependent manner. Thus, the glial determinant also regulates the efficiency of collective migration. NetrinB but not NetrinA serves as a chemoattractant and Unc5 contributes as a repellant Netrin receptor for glia migration. Our model includes strict spatial localization of a ligand, a cell autonomously acting receptor and a fate determinant that act coordinately to direct glia toward their final destination. DOI: http://dx.doi.org/10.7554/eLife.15983.001

Published
2016

44. Revisiting the role of the Gcm transcription factor, from master regulator to Swiss army knife

Author

Angela Giangrande, Pierre B. Cattenoz, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Cattenoz, Pierre

Subjects
0301 basic medicine, Nervous system, MESH: Signal Transduction, Embryo, Nonmammalian, MESH: Drosophila, MESH: Genes, Regulator, Cell, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Genes, Regulator, Drosophila Proteins, MESH: Animals, [SDV.BDD]Life Sciences [q-bio]/Development Biology, ComputingMilieux_MISCELLANEOUS, nervous system, JAK-STAT signaling pathway, Cell Differentiation, MESH: Transcription Factors, Cell biology, DNA-Binding Proteins, medicine.anatomical_structure, Drosophila, Signal Transduction, MESH: Cell Differentiation, Notch, hedgehog, MESH: Drosophila Proteins, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, stat, 03 medical and health sciences, Neuroblast, medicine, Animals, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, high-throughput screen, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Hedgehog, Transcription factor, Gene, Extra View, MESH: Embryo, Nonmammalian, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Gcm, JAK/STAT, immune system, 030104 developmental biology, Insect Science, Immunology, DamID, MESH: DNA-Binding Proteins, Transcription Factors
Abstract

International audience; Master genes are known to induce the differentiation of a multipotent cell into a specific cell type. These molecules are often transcription factors that switch on the regulatory cascade that triggers cell specification. Gcm was first described as the master gene of the glial fate in Drosophila as it induces the differentiation of neuroblasts into glia in the developing nervous system. Later on, Gcm was also shown to regulate the differentiation of blood, tendon and peritracheal cells as well as that of neuronal subsets. Thus, the glial master gene is used in at least 4 additional systems to promote differentiation. To understand the numerous roles of Gcm, we recently reported a genome-wide screen of Gcm direct targets in the Drosophila embryo. This screen provided new insight into the role and mode of action of this powerful transcription factor, notably on the interactions between Gcm and major differentiation pathways such as the Hedgehog, Notch and JAK/STAT. Here, we discuss the mode of action of Gcm in the different systems, we present new tissues that require Gcm and we revise the concept of 'master gene'.

Published
2016

45. Differing requirements for the Arabidopsis Rad51 paralogs in meiosis and DNA repair

Author

Bleuyard, Jean-Yves, Gallego, Maria, Savigny, Florence, White, Charles, Génétique, Reproduction et Développement (GReD), Centre National de la Recherche Scientifique (CNRS)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), and White, Charles

Subjects
animal structures, MESH: Mutation, DNA Repair, MESH: Sequence Homology, Amino Acid, Mitomycin, MESH: Cross-Linking Reagents, Molecular Sequence Data, Arabidopsis, [SDV.GEN] Life Sciences [q-bio]/Genetics, MESH: Amino Acid Sequence, MESH: Arabidopsis Proteins, MESH: Protein Isoforms, MESH: Phenotype, Protein Isoforms, MESH: Arabidopsis, MESH: Rad51 Recombinase, Amino Acid Sequence, MESH: Infertility, MESH: Phylogeny, Phylogeny, MESH: DNA Repair, [SDV.GEN]Life Sciences [q-bio]/Genetics, MESH: Molecular Sequence Data, Sequence Homology, Amino Acid, Arabidopsis Proteins, MESH: Mitomycin, fungi, MESH: Gamma Rays, DNA-Binding Proteins, Meiosis, enzymes and coenzymes (carbohydrates), MESH: Meiosis, Cross-Linking Reagents, Phenotype, Gamma Rays, Infertility, Mutation, Rad51 Recombinase, biological phenomena, cell phenomena, and immunity, MESH: DNA-Binding Proteins
Abstract

International audience; In addition to the recombinase Rad51, vertebrates have five paralogs of Rad51, all members of the Rad51-dependent recombination pathway. These paralogs form two complexes (Rad51C/Xrcc3 and Rad51B/C/D/Xrcc2), which play roles in somatic recombination, DNA repair and chromosome stability. However, little is known of their possible involvement in meiosis, due to the inviability of the corresponding knockout mice. We have recently reported that the Arabidopsis homolog of one of these Rad51 paralogs (AtXrcc3) is involved in DNA repair and meiotic recombination and present here Arabidopsis lines carrying mutations in three other Rad51 paralogs (AtRad51B, AtRad51C and AtXrcc2). Disruption of any one of these paralogs confers hypersensitivity to the DNA cross-linking agent Mitomycin C, but not to gamma-irradiation. Moreover, the atrad51c-1 mutant is the only one of these to show meiotic defects similar to those of the atxrcc3 mutant, and thus only the Rad51C/Xrcc3 complex is required to achieve meiosis. These results support conservation of functions of the Rad51 paralogs between vertebrates and plants and differing requirements for the Rad51 paralogs in meiosis and DNA repair.

Published
2016

46. Meiotic defects in the Arabidopsis rad50 mutant point to conservation of the MRX complex function in early stages of meiotic recombination

Author

Charles I. White, Maria Eugenia Gallego, Jean-Yves Bleuyard, Génétique, Reproduction et Développement (GReD), Centre National de la Recherche Scientifique (CNRS)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Clermont Auvergne [2017-2020] (UCA [2017-2020])-Centre National de la Recherche Scientifique (CNRS), and White, Charles

Subjects
0106 biological sciences, MESH: Mutation, Arabidopsis, [SDV.GEN] Life Sciences [q-bio]/Genetics, MESH: Arabidopsis Proteins, Biology, 01 natural sciences, Genetic recombination, Chromosomal crossover, 03 medical and health sciences, Meiosis, MRE11 Homologue Protein, Genetics, Homologous chromosome, MESH: Arabidopsis, Genetics (clinical), 030304 developmental biology, Recombination, Genetic, 0303 health sciences, [SDV.GEN]Life Sciences [q-bio]/Genetics, Arabidopsis Proteins, MESH: Fertility, biology.organism_classification, DNA-Binding Proteins, MESH: Meiosis, Fertility, MRX complex, Mutation, MESH: Recombination, Genetic, Homologous recombination, MESH: DNA-Binding Proteins, 010606 plant biology & botany
Abstract

International audience; The Rad50, Mre11 and Xrs2/Nbs1 proteins, which form the highly conserved MRX complex, perform a wide range of functions concerning the maintenance and function of DNA in eukaryotes. These include recombination, DNA repair, replication, telomere homeostasis and meiosis. Notwithstanding the attention paid to this complex, the inviability of vertebrate rad50 and mre11 mutants has led to a relative lack of information concerning the role of these proteins in meiosis in higher eukaryotes. We have previously reported that Arabidopsis atrad50 mutant plants are viable and that atrad50 mutant plants are sterile. The present study reports an analysis of the causes of this sterility and the implication of the AtRad50 protein in meiosis. Both male and female gametogenesis are defective in the Arabidopsis atrad50 mutant and cytological observation of male meiosis indicates that in the absence of the AtRad50 protein, homologous chromosomes are unable to synapse. Finally, the atrad50 mutation leads to the destruction of chromosomes during meiosis. These phenotypes support a role for the Arabidopsis MRX complex in early stages of meiotic recombination.

Published
2016

47. Germline RBBP6 mutations in familial myeloproliferative neoplasms

Author

Harutyunyan, A. S., Giambruno, R., Krendl, C., Stukalov, A., Klampfl, T., Berg, T., Chen, D., Feenstra, J. D. M., Jäger, R., Gisslinger, B., Gisslinger, H., Rumi, E., Passamonti, Francesco, Pietra, D., Müller, A. C., Parapatics, K., Breitwieser, F. P., Herrmann, R., Colinge, J., Bennett, K. L., Superti Furga, G., Cazzola, M., Hammond, E., Kralovics, R., Institut de Recherche en Cancérologie de Montpellier (IRCM - U1194 Inserm - UM), and CRLCC Val d'Aurelle - Paul Lamarque-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM)

Subjects
Male, Myeloproliferative Disorders, MESH: Humans, MESH: Pedigree, Ubiquitin-Protein Ligases, DNA Mutational Analysis, Letters to Blood, MESH: Carrier Proteins, MESH: Male, Pedigree, DNA-Binding Proteins, MESH: Germ-Line Mutation, Humans, Family, Female, MESH: DNA Mutational Analysis, Carrier Proteins, MESH: Family, MESH: Female, Germ-Line Mutation, ComputingMilieux_MISCELLANEOUS, MESH: DNA-Binding Proteins, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology, MESH: Myeloproliferative Disorders
Abstract

International audience

Published
2016

48. Systematic determination of transcription factor DNA-binding specificities in yeast

Author

Lourdes Peña-Castillo, Gwenael Badis, Memorial University of Newfoundland [St. John's], Génétique des Interactions macromoléculaires, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), G.B. work was supported by the CIHR, the Institut Pasteur and the Centre National pour la Recherche Scientifique. LPC’s work was supported by a NSERC Discovery Grant and Memorial University of Newfoundland., Frédéric Devaux, Memorial University of Newfoundland = Université Memorial de Terre-Neuve [St. John's, Canada] (MUN), Génétique des Interactions macromoléculaires / Genetics of Macromolecular Interactions, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Binding sites, [SDV]Life Sciences [q-bio], Regulome, Computational biology, Biology, DNA-binding protein, DNA binding domain, 03 medical and health sciences, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Enhancers, Transcription factors, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Binding site, Enhancer, Transcription factor, Gene, MESH: Protein Array Analysis, MESH: DNA, DNA-binding domain, Transcription regulation, cis-regulatory element, MESH: Transcription Factors, Molecular biology, MESH: Saccharomyces cerevisiae, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], 030104 developmental biology, MESH: Binding Sites, DNA microarray, MESH: DNA-Binding Proteins
Abstract

International audience; Understanding how genes are regulated, decoding their "regulome", is one of the main challenges of the post-genomic era. Here, we describe the in vitro method we used to associate cis-regulatory sites with cognate trans-regulators by characterizing the DNA-binding specificity of the vast majority of yeast transcription factors using Protein Binding Microarrays. This approach can be implemented to any given organism.

Published
2016

49. miR-9 Controls the Timing of Neurogenesis through the Direct Inhibition of Antagonistic Factors

Author

Øyvind Drivenes, Marion Coolen, Laure Bally-Cuif, Denis Thieffry, Thomas Becker, Neurobiologie et Développement (N&eD), Centre National de la Recherche Scientifique (CNRS), Institut de Neurobiologie Alfred Fessard (INAF), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Sars International Centre for Marine Molecular Biology, University of Bergen (UiB), Brain and Mind Research Institute, University of Technology Sydney (UTS), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Cell Differentiation, MESH: Alanine, Neurogenesis, MESH: Neurons, MESH: Zebrafish Proteins, MESH: Cell Cycle, ELAV-Like Protein 3, Hindbrain, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, 0302 clinical medicine, MESH: Basic Helix-Loop-Helix Transcription Factors, Basic Helix-Loop-Helix Transcription Factors, Animals, MESH: Animals, MESH: Zebrafish, Molecular Biology, Zebrafish, Gene, 030304 developmental biology, Progenitor, Neurons, 0303 health sciences, Alanine, biology, Cell Cycle, Embryogenesis, MESH: Hu Paraneoplastic Encephalomyelitis Antigens, Cell Differentiation, Azepines, Cell Biology, Anatomy, Zebrafish Proteins, biology.organism_classification, MESH: Neurogenesis, DNA-Binding Proteins, MicroRNAs, ELAV Proteins, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Ventricular zone, MESH: Azepines, MESH: MicroRNAs, Neuroscience, MESH: DNA-Binding Proteins, 030217 neurology & neurosurgery, Function (biology), Developmental Biology
Abstract

International audience; The timing of commitment and cell-cycle exit within progenitor populations during neurogenesis is a fundamental decision that impacts both the number and identity of neurons produced during development. We show here that microRNA-9 plays a key role in this process through the direct inhibition of targets with antagonistic functions. Across the ventricular zone of the developing zebrafish hindbrain, miR-9 expression occurs at a range of commitment stages. Abrogating miR-9 function transiently delays cell-cycle exit, leading to the increased generation of late-born neuronal populations. Target protection analyses in vivo identify the progenitor-promoting genes her6 and zic5 and the cell-cycle exit-promoting gene elavl3/HuC as sequential targets of miR-9 as neurogenesis proceeds. We propose that miR-9 activity generates an ambivalent progenitor state poised to respond to both progenitor maintenance and commitment cues, which may be necessary to adjust neuronal production to local extrinsic signals during late embryogenesis.

Published
2012
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50. CC2D1A Is a Regulator of ESCRT-III CHMP4B

Author

Heinrich G. Göttlinger, Charles Sabin, Bettina Hartlieb, Yoshiko Usami, Nolwenn Miguet, Sergiy V. Avilov, Winfried Weissenhorn, Nicolas Martinelli, Aurelien Dordor, Euripedes A. Ribeiro, Unit for Virus Host-Cell Interactions [Grenoble] (UVHCI), Centre National de la Recherche Scientifique (CNRS)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Université Joseph Fourier - Grenoble 1 (UJF), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Thomas, Frank

Subjects
Models, Molecular, Regulator, MESH: Protein Structure, Secondary, Plasma protein binding, Protein Structure, Secondary, MESH: Endosomal Sorting Complexes Required for Transport, MESH: HIV-1, MESH: Protein Structure, Tertiary, 0302 clinical medicine, Protein structure, Membrane fission, Structural Biology, Cell Line, Transformed, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 3. Good health, Transport protein, DNA-Binding Proteins, Protein Transport, Biochemistry, MESH: Repressor Proteins, MESH: HEK293 Cells, MESH: Models, Molecular, Protein Binding, MESH: Protein Transport, MESH: Mutation, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Endosome, Endosomes, Biology, Article, ESCRT, 03 medical and health sciences, Humans, MESH: Protein Binding, MESH: Cell Line, Transformed, Binding site, Molecular Biology, 030304 developmental biology, Binding Sites, MESH: Humans, Endosomal Sorting Complexes Required for Transport, Protein Structure, Tertiary, Repressor Proteins, HEK293 Cells, MESH: Binding Sites, MESH: Endosomes, Mutation, HIV-1, Biophysics, MESH: DNA-Binding Proteins, 030217 neurology & neurosurgery
Abstract

International audience; Endosomal sorting complexes required for transport (ESCRTs) regulate diverse processes ranging from receptor sorting at endosomes to distinct steps in cell division and budding of some enveloped viruses. Common to all processes is the membrane recruitment of ESCRT-III that leads to membrane fission. Here, we show that CC2D1A is a novel regulator of ESCRT-III CHMP4B function. We demonstrate that CHMP4B interacts directly with CC2D1A and CC2D1B with nanomolar affinity by forming a 1:1 complex. Deletion mapping revealed a minimal CC2D1A-CHMP4B binding construct, which includes a short linear sequence within the third DM14 domain of CC2D1A. The CC2D1A binding site on CHMP4B was mapped to the N-terminal helical hairpin. Based on a crystal structure of the CHMP4B helical hairpin, two surface patches were identified that interfere with CC2D1A interaction as determined by surface plasmon resonance. Introducing these mutations into a C-terminal truncation of CHMP4B that exerts a potent dominant negative effect on human immunodeficiency virus type 1 budding revealed that one of the mutants lost this effect completely. This suggests that the identified CC2D1A binding surface might be required for CHMP4B polymerization, which is consistent with the finding that CC2D1A binding to CHMP4B prevents CHMP4B polymerization in vitro. Thus, CC2D1A might act as a negative regulator of CHMP4B function.

Published
2012

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