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1. Rouse model with transient intramolecular contacts on a timescale of seconds recapitulates folding and fluctuation of yeast chromosomes

Author

Kerstin Bystricky, Aurélien Bancaud, Renjie Wang, Jean-Marc Victor, Cédric Vaillant, Marius Socol, Pascal Carrivain, Daniel Jost, Olivier Gadal, Christophe Normand, Eric Le Cam, Vincent Dahirel, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Équipe Micro-Nanofluidique pour les sciences de la vie et de l’environnement (LAAS-MILE), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT), Laboratoire de biologie moléculaire eucaryote (LBME), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Henan University of Science and Technology, Biologie Computationnelle et Mathématique (TIMC-IMAG-BCM), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de Physique de l'ENS Lyon (Phys-ENS), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), PHysicochimie des Electrolytes et Nanosystèmes InterfaciauX (PHENIX), Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Nutrition et Alimentation des Populations aux Suds (NutriPass), Université Montpellier 1 (UM1)-Institut de Recherche pour le Développement (IRD)-Université Montpellier 2 - Sciences et Techniques (UM2)-Université de Montpellier (UM)-Institut national d’études supérieures agronomiques de Montpellier (Montpellier SupAgro), Laboratoire de Physique Théorique de la Matière Condensée (LPTMC), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire des matériaux et du génie physique (LMGP), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique de Grenoble (INPG), Université Toulouse III - Paul Sabatier (UPS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes (UGA), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université Paris-Saclay, Université Pierre et Marie Curie - Paris 6 (UPMC)-ESPCI ParisTech-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UPS), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Université Toulouse 1 Capitole (UT1), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), École normale supérieure - Lyon (ENS Lyon)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), and Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)

Subjects
[PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], DNA Folding, Chromatin and Epigenetics, Chromosome conformation capture, 03 medical and health sciences, Motion, 0302 clinical medicine, Genetics, Nucleosome, Gene Regulation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, DNA, Fungal, 030304 developmental biology, 0303 health sciences, biology, Models, Genetic, Dynamics (mechanics), Gene regulation, Chromatin and Epigenetics, Chromatin, Nucleosomes, Folding (chemistry), Kinetics, Histone, Chemical physics, biology.protein, Chromosomes, Fungal, Parametrization, 030217 neurology & neurosurgery
Abstract

DNA folding and dynamics along with major nuclear functions are determined by chromosome structural properties, which remain, thus far, elusive in vivo. Here, we combine polymer modeling and single particle tracking experiments to determine the physico-chemical parameters of chromatin in vitro and in living yeast. We find that the motion of reconstituted chromatin fibers can be recapitulated by the Rouse model using mechanical parameters of nucleosome arrays deduced from structural simulations. Conversely, we report that the Rouse model shows some inconsistencies to analyze the motion and structural properties inferred from yeast chromosomes determined with chromosome conformation capture techniques (specifically, Hi-C). We hence introduce the Rouse model with Transient Internal Contacts (RouseTIC), in which random association and dissociation occurs along the chromosome contour. The parametrization of this model by fitting motion and Hi-C data allows us to measure the kinetic parameters of the contact formation reaction. Chromosome contacts appear to be transient; associated to a lifetime of seconds and characterized by an attractive energy of –0.3 to –0.5 kBT. We suggest attributing this energy to the occurrence of histone tail-DNA contacts and notice that its amplitude sets chromosomes in ‘theta’ conditions, in which they are poised for compartmentalization and phase separation.

Published
2019
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2. Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

Author

Christophe Dez, Carlos Fernández-Tornero, Isabelle Léger-Silvestre, Marie-Kerguelen Sarthou, Michael Pilsl, Anthony K. Henras, Olga Calvo, Olivier Gadal, Marta Kwapisz, Christophe Normand, Sylvain Audibert, Titouan Genty, Adrien Chauvier, Tommy Darrière, Herbert Tschochner, Agencia Estatal de Investigación (España), Agence Nationale de la Recherche (France), Université de Toulouse, Ministerio de Economía y Competitividad (España), Fundación Ramón Areces, Ministerio de Ciencia, Innovación y Universidades (España), Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Universität Regensburg, Biochemie-Zentrum Regensburg (BZR), Lehrstuhl Biochemie III, Regensburg, Germany, Département de biologie [Sherbrooke] (UdeS), Faculté des sciences [Sherbrooke] (UdeS), Université de Sherbrooke (UdeS)-Université de Sherbrooke (UdeS), Instituto de Biología Funcional y Genómica, IBFG-CSIC, Universidad de Salamanca, Salamanca, Spain, and Centro de Investigaciones Biológicas, CSIC, Madrid, Spain

Subjects
Cancer Research, Heredity, Transcription, Genetic, viruses, Mutant, QH426-470, Biochemistry, Polymerases, Suppressor Genes, chemistry.chemical_compound, Genetic Suppression, 0302 clinical medicine, Transcription (biology), RNA Polymerase I, RNA polymerase, RNA Precursors, Super-active mutant, ComputingMilieux_MISCELLANEOUS, Genetics (clinical), Polymerase, Gel Electrophoresis, 0303 health sciences, biology, RNA Polymerase, Eukaryota, Cell biology, Nucleic acids, Ribosomal RNA, Rpa49, Pol1 Transcription Initiation Complex Proteins, Research Article, Cellular structures and organelles, Saccharomyces cerevisiae Proteins, Saccharomyces cerevisiae, Research and Analysis Methods, DNA, Ribosomal, 03 medical and health sciences, Electrophoretic Techniques, Genetic, ARN polimerasa I, Gene Types, DNA-binding proteins, RNA polymerase I, Genetics, Point Mutation, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Non-coding RNA, Gene, Molecular Biology, Transcription factor, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, [SDV.GEN]Life Sciences [q-bio]/Genetics, Biology and life sciences, 2409 Genética, 3201.02 Genética Clínica, Organisms, Fungi, RNA, Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Yeast, chemistry, RNA, Ribosomal, Enzyme, Mutation, Biochemical analysis, biology.protein, Ribosomes, 030217 neurology & neurosurgery, DNA, Transcription Factors
Abstract

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that wild-type RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I., This work was supported by the Agence Nationale de la Recherche (ANDY) and the IDEX of Toulouse University (Clemgene and Nudgene). We thank Margaux Cescato, Lilian Guillot and Jorge Perez-Fernandez for strain and plasmid constructions. YCp50-26 carrying RPA49 was kindly provided by P. Thuriaux. We also thank Melanie Panarotto for the Miller spread analysis. This work benefited from exchanges with all unit members, including Anthony Henras, Frederic Beckouët, Lise Dauban, and Marta Kwapisz. We thank Stephanie Balor and Vanessa Soldan from the METI platform for the TEM acquisitions. CFT was supported by grants BFU2013-48374-P and BFU2017-87397-P of the Spanish Ministry of Economy and Competitiveness, and by the Ramón Areces Foundation.

Published
2019
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3. In vivo, chromatin is a fluctuating polymer chain at equilibrium constrained by internal friction

Author

Marius Socol, Kerstin Bystricky, Zedek A, Jean-Marc Victor, Daniel Jost, Renjie Wang, Olivier Gadal, Christophe Normand, Aurélien Bancaud, Dahirel, and Pascal Carrivain

Subjects
Genetics, 0303 health sciences, DNA Folding, Biology, 01 natural sciences, Chromatin, Chromosome conformation capture, 03 medical and health sciences, Match moving, Transcription (biology), 0103 physical sciences, Biophysics, Kuhn length, Nucleosome, 010306 general physics, 030304 developmental biology, Chromatin Fiber
Abstract

Chromosome mechanical properties determine DNA folding and dynamics, and underlie all major nuclear functions. Here we combine modeling and real-time motion tracking experiments to infer the physical parameters describing chromatin fibers. In vitro, motion of nucleosome arrays can be accurately modeled by assuming a Kuhn length of 35-55 nm. In vivo, the amplitude of chromosome fluctuations is drastically reduced, and depends on transcription. Transcription activation increases chromatin dynamics only if it involves gene relocalization, while global transcriptional inhibition augments the fluctuations, yet without relocalization. Chromatin fiber motion is accounted for by a model of equilibrium fluctuations of a polymer chain, in which random contacts along the chromosome contour induce an excess of internal friction. Simulations that reproduce chromosome conformation capture and imaging data corroborate this hypothesis. This model unravels the transient nature of chromosome contacts, characterized by a life time of ∼2 seconds and a free energy of formation of ∼1 kBT.

Published
2017
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4. Structure-function analysis of hmo1 unveils an ancestral organization of hmg-box factors involved in ribosomal dna transcription from yeast to human

Author

Isabelle Léger-Silvestre, Axel B. Berger, Jorge Perez-Fernandez, Christine Colleran, Christophe Normand, Olivier Gadal, Benjamin Albert, Brian McStay, and Christophe Dez

Subjects
Ribosomal Proteins, Saccharomyces cerevisiae Proteins, Transcription, Genetic, Amino Acid Motifs, 5.8S ribosomal RNA, Ribosome biogenesis, Saccharomyces cerevisiae, Gene Regulation, Chromatin and Epigenetics, Biology, DNA, Ribosomal, rdna transcription, Structure-Activity Relationship, 03 medical and health sciences, 5S ribosomal RNA, RNA Polymerase I, Ribosomal protein, HMGB Proteins, rna-polymerase-i, Genetics, RNA polymerase I, Humans, Amino Acid Sequence, subunit, Ribosomal DNA, Conserved Sequence, 030304 developmental biology, gene-transcription, 0303 health sciences, nucleolar transcription, protein genes, factor ubf, 030302 biochemistry & molecular biology, High Mobility Group Proteins, Promoter, Ribosomal RNA, fission yeast, Protein Structure, Tertiary, saccharomyces-cerevisiae, Schizosaccharomyces pombe Proteins, Pol1 Transcription Initiation Complex Proteins, Cell Nucleolus, upstream binding-factor
Abstract

Ribosome biogenesis is a major metabolic effort for growing cells. In Saccharomyces cerevisiae, Hmo1, an abundant high-mobility group box protein (HMGB) binds to the coding region of the RNA polymerase I transcribed ribosomal RNAs genes and the promoters of similar to 70% of ribosomal protein genes. In this study, we have demonstrated the functional conservation of eukaryotic HMGB proteins involved in ribosomal DNA (rDNA) transcription. We have shown that when expressed in budding yeast, human UBF1 and a newly identified Sp-Hmo1 (Schizosaccharomyces pombe) localize to the nucleolus and suppress growth defect of the RNA polymerase I mutant rpa49-Delta. Owing to the multiple functions of both proteins, Hmo1 and UBF1 are not fully interchangeable. By deletion and domains swapping in Hmo1, we identified essential domains that stimulate rDNA transcription but are not fully required for stimulation of ribosomal protein genes expression. Hmo1 is organized in four functional domains: a dimerization module, a canonical HMGB motif followed by a conserved domain and a C-terminal nucleolar localization signal. We propose that Hmo1 has acquired species-specific functions and shares with UBF1 and Sp-Hmo1 an ancestral function to stimulate rDNA transcription.

Published
2013

5. Systematic characterization of the conformation and dynamics of budding yeast chromosome XII

Author

Purnima Bhargava, Ashutosh Shukla, Aurélien Bancaud, Olivier Gadal, Benjamin Albert, Hua Wong, Alain Kamgoue, Hicham Saad, Julien Mathon, Christophe Zimmer, Christophe Normand, Julien Mozziconacci, David Villa, Isabelle Léger-Silvestre, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées, Council of Scientific and Industrial Research [India] (CSIR), Laboratoire de Physique Théorique de la Matière Condensée (LPTMC), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC), GDR 3536 Architecture et Dynamique Nucléaires (ADN), Université Pierre et Marie Curie - Paris 6 (UPMC), Imagerie et Modélisation, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), This work was supported by an ATIP-plus grant from Centre National de la Recherche Scientifique, by the Agence Nationale de la Recherche (Nucleopol, Ribeuc, and program JCJC), Jeune équipe from FRM, and University Paul Sabatier (a fellowship for J. Mathon). This work is a French-Indian collaborative effort funded by Indo-French Centre for the Promotion of Advanced Research (IFCPAR) Project 4103 between the laboratories of Dr. Bhargava and Dr. Gadal., ANR-08-PCVI-0036,NUCLEOPOL,Integrated structural biology of rDNA transcription(2008), ANR-06-BLAN-0037,RIBEUC,Ribosome biogenesis in eukaryotes: mechanisms and connections with other pathways(2006), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie moléculaire eucaryote du CNRS (LBME), Université Paul Sabatier - Toulouse 3 (UPS) - Centre National de la Recherche Scientifique (CNRS), Laboratoire d'analyse et d'architecture des systèmes [Toulouse] (LAAS), Institut National Polytechnique [Toulouse] (INP) - Institut National des Sciences Appliquées - Toulouse (INSA Toulouse) - Université Paul Sabatier - Toulouse 3 (UPS) - Centre National de la Recherche Scientifique (CNRS), Council of Scientific & Industrial Research [India] (CSIR), Université Pierre et Marie Curie - Paris 6 (UPMC) - Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] - Centre National de la Recherche Scientifique (CNRS), ANR-08-PCVI-0036, NUCLEOPOL, Integrated structural biology of rDNA transcription(2008), and ANR-06-BLAN-0037, RIBEUC, Ribosome biogenesis in eukaryotes: mechanisms and connections with other pathways(2006)

Subjects
Transcription, Genetic, Nucleolus, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Saccharomyces cerevisiae, Genome, DNA, Ribosomal, Time-Lapse Imaging, 03 medical and health sciences, 0302 clinical medicine, Report, Computer Simulation, DNA, Fungal, Ribosomal DNA, Research Articles, 030304 developmental biology, Genetics, Sirolimus, 0303 health sciences, biology, [PHYS.PHYS.PHYS-BIO-PH] Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chromosome, Chromosome Mapping, Cell Biology, Telomere, biology.organism_classification, Chromatin, High-Throughput Screening Assays, Saccharomycetales, Genetic Loci, Chromosomes, Fungal, Genome, Fungal, 030217 neurology & neurosurgery, Cell Nucleolus
Abstract

Comprehensive analysis of the intranuclear territories and motion of budding yeast chromosome XII loci suggests that long-range chromosome architecture is mainly determined by the physical principles of polymers., Chromosomes architecture is viewed as a key component of gene regulation, but principles of chromosomal folding remain elusive. Here we used high-throughput live cell microscopy to characterize the conformation and dynamics of the longest chromosome of Saccharomyces cerevisiae (XII). Chromosome XII carries the ribosomal DNA (rDNA) that defines the nucleolus, a major hallmark of nuclear organization. We determined intranuclear positions of 15 loci distributed every ∼100 kb along the chromosome, and investigated their motion over broad time scales (0.2–400 s). Loci positions and motions, except for the rDNA, were consistent with a computational model of chromosomes based on tethered polymers and with the Rouse model from polymer physics, respectively. Furthermore, rapamycin-dependent transcriptional reprogramming of the genome only marginally affected the chromosome XII internal large-scale organization. Our comprehensive investigation of chromosome XII is thus in agreement with recent studies and models in which long-range architecture is largely determined by the physical principles of tethered polymers and volume exclusion.

Published
2013
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6. Nuclear organization and chromatin dynamics in yeast: Biophysical models or biologically driven interactions?

Author

Christophe Normand, Isabelle Léger-Silvestre, Olivier Gadal, Benjamin Albert, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Biologie Cellulaire du Noyau (BCN), and Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)

Subjects
Saccharomyces cerevisiae, Biophysics, Computational biology, Biochemistry, Genome, Biophysical Phenomena, Chromosomes, Diffusion, 03 medical and health sciences, 0302 clinical medicine, Structural Biology, Gene Expression Regulation, Fungal, Genetics, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Epigenetics, Nuclear pore, Scaffold/matrix attachment region, Molecular Biology, Gene, 030304 developmental biology, Cell Nucleus, 0303 health sciences, biology, Models, Theoretical, Chromatin Assembly and Disassembly, biology.organism_classification, Chromatin, Nuclear transport, 030217 neurology & neurosurgery
Abstract

International audience; Over the past decade, tremendous progress has been made in understanding the spatial organization of genes and chromosomes. Nuclear organization can be thought of as information that is not encoded in DNA, but which nevertheless impacts gene expression. Nuclear organizational influences can be cell-specific and are potentially heritable. Thus, nuclear organization fulfills all the criteria necessary for it to be considered an authentic level of epigenetic information. Chromosomal nuclear organization is primarily dictated by the biophysical properties of chromatin. Diffusion models of polymers confined in the crowded nuclear space accurately recapitulate experimental observation. Diffusion is a Brownian process, which implies that the positions of chromosomes and genes are not defined deterministically but are likely to be dictated by the laws of probability. Despite the small size of their nuclei, budding yeast have been instrumental in discovering how epigenetic information is encoded in the spatial organization of the genome. The relatively simple organization of the yeast nucleus and the very high number of genetically identical cells that can be observed under fluorescent microscopy allow statistically robust definitions of the gene and chromosome positions in the nuclear space to be constructed. In this review, we will focus on how the spatial organization of the chromatin in the yeast nucleus might impact transcription. This article is part of a Special Issue entitled: Nuclear Transport and RNA Processing.

Published
2012
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7. RNA polymerase I-specific subunits promote polymerase clustering to enhance the rRNA gene transcription cycle

Author

Christophe Normand, Isabelle Léger-Silvestre, Benjamin Albert, Jorge Perez-Fernandez, Kostya I. Panov, Joost C. B. M. Zomerdijk, Patrick Schultz, Martin K. Ostermaier, Olivier Gadal, Laboratoire de biologie moléculaire eucaryote (LBME), Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)

Subjects
Transcription, Genetic, MESH: RNA Polymerase I, viruses, Nuclear Localization Signals, RNA polymerase II, MESH: Nuclear Localization Signals, 0302 clinical medicine, RNA Polymerase I, Gene Expression Regulation, Fungal, Transcriptional regulation, Polymerase, Research Articles, Conserved Sequence, 0303 health sciences, MESH: Genetic Complementation Test, MESH: Conserved Sequence, biology, MESH: Protein Multimerization, MESH: Cell Nucleus Shape, MESH: Transcription Factors, MESH: Protein Subunits, MESH: Saccharomyces cerevisiae, MESH: Schizosaccharomyces, MESH: Cell Nucleolus, Cell Nucleolus, MESH: Gene Expression Regulation, Fungal, Cell Nucleus Shape, Saccharomyces cerevisiae, RNA polymerase III, Article, 03 medical and health sciences, Schizosaccharomyces, MESH: Genes, rRNA, RNA polymerase I, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, MESH: Humans, MESH: Transcription, Genetic, Genetic Complementation Test, Genes, rRNA, Cell Biology, Processivity, Ribosomal RNA, Molecular biology, Protein Subunits, biology.protein, DNA polymerase I, Protein Multimerization, 030217 neurology & neurosurgery, Transcription Factors
Abstract

The Pol I subunits Rpa34 and Rpa49, required for elongation and 35S rRNA production, promote clustering of neighboring Pol I complexes to enhance the rRNA gene transcription cycle., RNA polymerase I (Pol I) produces large ribosomal RNAs (rRNAs). In this study, we show that the Rpa49 and Rpa34 Pol I subunits, which do not have counterparts in Pol II and Pol III complexes, are functionally conserved using heterospecific complementation of the human and Schizosaccharomyces pombe orthologues in Saccharomyces cerevisiae. Deletion of RPA49 leads to the disappearance of nucleolar structure, but nucleolar assembly can be restored by decreasing ribosomal gene copy number from 190 to 25. Statistical analysis of Miller spreads in the absence of Rpa49 demonstrates a fourfold decrease in Pol I loading rate per gene and decreased contact between adjacent Pol I complexes. Therefore, the Rpa34 and Rpa49 Pol I–specific subunits are essential for nucleolar assembly and for the high polymerase loading rate associated with frequent contact between adjacent enzymes. Together our data suggest that localized rRNA production results in spatially constrained rRNA production, which is instrumental for nucleolar assembly.

Published
2011
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8. Analysis of the binding of the N-terminal conserved domain of yeast Cbf5p to a box H/ACA snoRNA

Author

Annie Mougin, Christophe Normand, Michèle Caizergues-Ferrer, Sophie Quevillon-Cheruel, Régine Capeyrou, Yves Henry, Laboratoire de biologie moléculaire eucaryote (LBME), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), and CNRS, UPS, LNCC, ANR

Subjects
Saccharomyces cerevisiae Proteins, Molecular Sequence Data, Ribosome biogenesis, Saccharomyces cerevisiae, box H/ACA snoRNA, Biology, Dyskerin, Article, Conserved sequence, 03 medical and health sciences, 0302 clinical medicine, Ribonucleases, RNA, Small Nucleolar, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Guide RNA, Amino Acid Sequence, Protein Footprinting, Small nucleolar RNA, Molecular Biology, rRNA pseudouridylation, Conserved Sequence, Hydro-Lyases, 030304 developmental biology, Ribonucleoprotein, 2. Zero hunger, 0303 health sciences, Binding Sites, Base Sequence, Sequence Homology, Amino Acid, Protein footprinting, yeast Cbf5p, RNA, RNA, Fungal, Ribonucleoproteins, Small Nuclear, Molecular biology, Recombinant Proteins, Protein Structure, Tertiary, Biochemistry, RNA–protein interactions, Nucleic Acid Conformation, box H/ACA snoRNP, Microtubule-Associated Proteins, 030217 neurology & neurosurgery, Protein Binding
Abstract

During ribosome biogenesis, the RNA precursor to mature rRNAs undergoes numerous post-transcriptional chemical modifications of bases, including conversions of uridines to pseudouridines. In archaea and eukaryotes, these conversions are performed by box H/ACA small ribonucleoprotein particles (box H/ACA RNPs), which contain a small guide RNA responsible for the selection of substrate uridines and four proteins, including the pseudouridine synthase, Cbf5p. So far, no in vitro reconstitution of eukaryotic box H/ACA RNPs from purified components has been achieved, principally due to difficulties in purifying recombinant eukaryotic Cbf5p. In this study, we present the purification of a truncated derivative of yeast Cbf5p (Cbf5Δp) that retains the highly conserved TRUB and PUA domains. We have used band retardation assays to show that Cbf5Δp on its own binds to box H/ACA small nucleolar (sno)RNAs. We demonstrate that the conserved H and ACA boxes enhance the affinity of the protein for the snoRNA. Furthermore, like its archaeal homologs, Cbf5Δp can bind to a single stem–loop-box ACA RNA. Finally, we report the first enzymatic footprinting analysis of a Cbf5–RNA complex. Our results are compatible with the view that two molecules of Cbf5p interact with a binding platform constituted by the 5′ end of the RNA, the single-stranded hinge domain containing the conserved H box, and the 3′ end of the molecule, including the conserved ACA box.

Published
2006
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