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Home > Scientific Publications

Genome Biology Department Publications

2017


  • A. F. Amorim, D. Pinto, L. Kuras, and L. Fernandes, “Absence of Gim proteins, but not GimC complex, alter stress-induced transcription”, Biochimica Et Biophysica Acta, 2017.
    Abstract: Saccharomyces cerevisiae GimC (mammalian Prefoldin) is a hexameric (Gim1-6) cytoplasmic complex involved in the folding pathway of actin/tubulin. In contrast to a shared role in GimC complex, we show that absence of individual Gim proteins results in distinct stress responses. No concomitant alteration in F-actin integrity was observed. Transcription of stress responsive genes is altered in gim2Δ, gim3Δ and gim6Δ mutants: TRX2 gene is induced in these mutants but with a profile diverging from type cells, whereas CTT1 and HSP26 fail to be induced. Remaining gimΔ mutants display stress transcript abundance comparable to wild type cells. No alteration in the nuclear localization of the transcriptional activators for TRX2 (Yap1) and CTT1/HSP26 (Msn2) was observed in gim2Δ. In accordance with TRX2 induction, RNA polymerase II occupancy at TRX2 discriminates the wild type from gim2Δ and gim6Δ. In contrast, RNA polymerase II occupancy at CTT1 is similar in wild type and gim2Δ, but higher in gim6Δ. The absence of active RNA polymerase II at CTT1 in gim2Δ, but not in wild type and gim1Δ, explains the respective CTT1 transcript outputs. Altogether our results put forward the need of Gim2, Gim3 and Gim6 in oxidative and osmotic stress activated transcription; others Gim proteins are dispensable. Consequently, the participation of Gim proteins in activated-transcription is independent from the GimC complex.
    Tags: DBG, Gim proteins, PEPS, stress, Transcription regulation.

  • O. Arnaiz, E. Van Dijk, M. Bétermier, M. Lhuillier-Akakpo, A. de Vanssay, S. Duharcourt, E. Sallet, J. Gouzy, and L. Sperling, “Improved methods and resources for paramecium genomics: transcription units, gene annotation and gene expression”, BMC genomics, vol. 18, no. 1, p. 483, 2017.
    Abstract: BACKGROUND: The 15 sibling species of the Paramecium aurelia cryptic species complex emerged after a whole genome duplication that occurred tens of millions of years ago. Given extensive knowledge of the genetics and epigenetics of Paramecium acquired over the last century, this species complex offers a uniquely powerful system to investigate the consequences of whole genome duplication in a unicellular eukaryote as well as the genetic and epigenetic mechanisms that drive speciation. High quality Paramecium gene models are important for research using this system. The major aim of the work reported here was to build an improved gene annotation pipeline for the Paramecium lineage. RESULTS: We generated oriented RNA-Seq transcriptome data across the sexual process of autogamy for the model species Paramecium tetraurelia. We determined, for the first time in a ciliate, candidate P. tetraurelia transcription start sites using an adapted Cap-Seq protocol. We developed TrUC, multi-threaded Perl software that in conjunction with TopHat mapping of RNA-Seq data to a reference genome, predicts transcription units for the annotation pipeline. We used EuGene software to combine annotation evidence. The high quality gene structural annotations obtained for P. tetraurelia were used as evidence to improve published annotations for 3 other Paramecium species. The RNA-Seq data were also used for differential gene expression analysis, providing a gene expression atlas that is more sensitive than the previously established microarray resource. CONCLUSIONS: We have developed a gene annotation pipeline tailored for the compact genomes and tiny introns of Paramecium species. A novel component of this pipeline, TrUC, predicts transcription units using Cap-Seq and oriented RNA-Seq data. TrUC could prove useful beyond Paramecium, especially in the case of high gene density. Accurate predictions of 3' and 5' UTR will be particularly valuable for studies of gene expression (e.g. nucleosome positioning, identification of cis regulatory motifs). The P. tetraurelia improved transcriptome resource, gene annotations for P. tetraurelia, P. biaurelia, P. sexaurelia and P. caudatum, and Paramecium-trained EuGene configuration are available through ParameciumDB ( http://paramecium.i2bc.paris-saclay.fr ). TrUC software is freely distributed under a GNU GPL v3 licence ( https://github.com/oarnaiz/TrUC ).
    Tags: ANGE, Autogamy, Cap-Seq, Ciliate, DBG, Differential gene expression, Gene annotation, MICMAC, RNA-Seq, TSS.


  • S. Barral, Y. Morozumi, H. Tanaka, E. Montellier, J. Govin, M. de Dieuleveult, G. Charbonnier, Y. Couté, D. Puthier, T. Buchou, F. Boussouar, T. Urahama, F. Fenaille, S. Curtet, P. Héry, N. Fernandez-Nunez, H. Shiota, M. Gérard, S. Rousseaux, H. Kurumizaka, and S. Khochbin, “Histone Variant H2A.L.2 Guides Transition Protein-Dependent Protamine Assembly in Male Germ Cells”, Molecular Cell, vol. 66, no. 1, p. 89-101.e8, 2017.

  • S. Berlivet, I. Hmitou, H. Picaud, and M. Gérard, “Efficient Depletion of Essential Gene Products for Loss-of-Function Studies in Embryonic Stem Cells”, Methods in Molecular Biology (Clifton, N.J.), vol. 1622, p. 91-100, 2017.
    Abstract: The development of the CRISPR/Cas9 technology has provided powerful methods to target genetic alterations. However, investigating the function of genes essential for cell survival remains problematic, because genetic ablation of these genes results in cell death. As a consequence, cells recombined at the targeted gene and fully depleted of the gene product cannot be obtained. RNA interference is well suited for the study of essential genes, but this approach often results in a partial depletion of the targeted gene product, which can lead to misinterpretations. We previously developed the pHYPER shRNA vector, a high efficiency RNA interference vector, which is based on a 2.5-kb mouse genomic fragment encompassing the H1 gene. We provide here a pHYPER-based protocol optimized to study the function of essential gene products in mouse embryonic stem cells.
    Tags: DBG, Electroporation, Embryonic stem cell, Essential genes, pHYPER, Puromycin selection, REMOD, RNA Interference, shRNA.

  • L. Bidou, O. Bugaud, V. Belakhov, T. Baasov, and O. Namy, “Characterization of new-generation aminoglycoside promoting premature termination codon readthrough in cancer cells”, RNA biology, p. 1-11, 2017.
    Abstract: Nonsense mutations, generating premature termination codons (PTCs), account for 10% to 30% of the mutations in tumor suppressor genes. Nonsense translational suppression, induced by small molecules including gentamicin and G418, has been suggested as a potential therapy to counteract the deleterious effects of nonsense mutations in several genetic diseases and cancers. We describe here that NB124, a synthetic aminoglycoside derivative recently developed especially for PTC suppression, strongly induces apoptosis in human tumor cells by promoting high level of PTC readthrough. Using a reporter system, we showed that NB124 suppressed several of the PTCs encountered in tumor suppressor genes, such as the p53 and APC genes. We also showed that NB124 counteracted p53 mRNA degradation by nonsense-mediated decay (NMD). Both PTC suppression and mRNA stabilization contributed to the production of a full-length p53 protein capable of activating p53-dependent genes, thereby specifically promoting high levels of apoptosis. This new-generation aminoglycoside thus outperforms the only clinically available readthrough inducer (gentamicin). These results have important implications for the development of personalised treatments of PTC-dependent diseases and for the development of new drugs modifying translation fidelity.
    Tags: Aminoglycoside, Apoptosis, cancer, DBG, GST, p53, stop codon readthrough.


  • W. V. Bienvenut, J. - P. Scarpelli, J. Dumestier, T. Meinnel, and C. Giglione, “EnCOUNTer: a parsing tool to uncover the mature N-terminus of organelle-targeted proteins in complex samples”, BMC Bioinformatics, vol. 18, no. 1, 2017.

  • M. Boudard, D. Barth, J. Bernauer, A. Denise, and J. Cohen, “GARN2: coarse-grained prediction of 3D structure of large RNA molecules by regret minimization”, Bioinformatics (Oxford, England), 2017.
    Abstract: Motivation: Predicting the 3D structure of RNA molecules is a key feature towards predicting their functions. Methods which work at atomic or nucleotide level are not suitable for large molecules. In these cases, coarse-grained prediction methods aim to predict a shape which could be refined later by using more precise methods on smaller parts of the molecule. Results: We developed a complete method for sampling 3D RNA structure at a coarse-grained model, taking a secondary structure as input. One of the novelties of our method is that a second step extracts two best possible structures close to the native, from a set of possible structures. Although our method benefits from the first version of GARN, some of the main features on GARN2 are very different. GARN2 is much faster than the previous version and than the well-known methods of the state-of-art. Our experiments show that GARN2 can also provide better structures than the other state-of-the-art methods. Availability and implementations: GARN2 is written in Java. It is freely distributed and available at: http://garn.lri.fr/ . Contacts: melanie.boudard@lri.fr , johanne.cohen@lri.fr. Supplementary information: Supplementary data are available at Bioinformatics online.
    Tags: BIM, DBG.

  • C. Bouthier de la Tour, M. Mathieu, L. Meyer, P. Dupaigne, F. Passot, P. Servant, S. Sommer, E. Le Cam, and F. Confalonieri, “In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium”, PloS One, vol. 12, no. 5, p. e0177751, 2017.
    Abstract: The bacterium Deinococcus radiodurans possesses a set of Deinococcus-specific genes highly induced after DNA damage. Among them, ddrC (dr0003) was recently re-annotated, found to be in the inverse orientation and called A2G07_00380. Here, we report the first in vivo and in vitro characterization of the corrected DdrC protein to better understand its function in irradiated cells. In vivo, the ΔddrC null mutant is sensitive to high doses of UV radiation and the ddrC deletion significantly increases UV-sensitivity of ΔuvrA or ΔuvsE mutant strains. We show that the expression of the DdrC protein is induced after γ-irradiation and is under the control of the regulators, DdrO and IrrE. DdrC is rapidly recruited into the nucleoid of the irradiated cells. In vitro, we show that DdrC is able to bind single- and double-stranded DNA with a preference for the single-stranded DNA but without sequence or shape specificity and protects DNA from various nuclease attacks. DdrC also condenses DNA and promotes circularization of linear DNA. Finally, we show that the purified protein exhibits a DNA strand annealing activity. Altogether, our results suggest that DdrC is a new DNA binding protein with pleiotropic activities. It might maintain the damaged DNA fragments end to end, thus limiting their dispersion and extensive degradation after exposure to ionizing radiation. DdrC might also be an accessory protein that participates in a single strand annealing pathway whose importance in DNA repair becomes apparent when DNA is heavily damaged.
    Tags: DBG, RBA.

  • P. Brézellec, M. - A. Petit, S. Pasek, I. Vallet-Gely, C. Possoz, and J. - L. Ferat, “Domestication of Lambda Phage Genes into a Putative Third Type of Replicative Helicase Matchmaker”, Genome Biology and Evolution, vol. 9, no. 6, p. 1561-1566, 2017.
    Abstract: At the onset of the initiation of chromosome replication, bacterial replicative helicases are recruited and loaded on the DnaA-oriC nucleoprotein platform, assisted by proteins like DnaC/DnaI or DciA. Two orders of bacteria appear, however, to lack either of these factors, raising the question of the essentiality of these factors in bacteria. Through a phylogenomic approach, we identified a pair of genes that could have substituted for dciA. The two domesticated genes are specific of the dnaC/dnaI- and dciA-lacking organisms and apparently domesticated from lambdoid phage genes. They derive from λO and λP and were renamed dopC and dopE, respectively. DopE is expected to bring the replicative helicase to the bacterial origin of replication, while DopC might assist DopE in this function. The confirmation of the implication of DopCE in the handling of the replicative helicase at the onset of replication in these organisms would generalize to all bacteria and therefore to all living organisms the need for specific factors dedicated to this function.
    Tags: DBG, dciA, dnaC, EMC2, lambda phage, OCB, replication initiation, replicative helicase, viral gene domestication.

  • O. Bugaud, N. Barbier, H. Chommy, N. Fiszman, A. Le Gall, D. Dullin, M. Saguy, N. Westbrook, K. Perronet, and O. Namy, “Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single molecule fluorescence microscopy”, RNA (New York, N.Y.), 2017.
    Abstract: Protein synthesis is a complex multi-step process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labelling of the ribosomes are much more complicated. Here we use a non-canonical translation initiation based on internal ribosome entry sites (IRES) and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA. We explore initiation by two types of IRES, the intergenic IRES of Cricket Paralysis virus (CrPV) and the hepatitis C (HCV) IRES, and show that they both strongly limit the rate of the first elongation steps compared to the following ones suggesting that those first elongation cycles do not correspond to a canonical elongation. This new system opens the possibility to study both IRES-mediated initiation and elongation kinetics of eukaryotic translation and will undoubtedly be a valuable tool to investigate the role of translation machinery modifications in human diseases.
    Tags: DBG, Eukaryotic translation, GST, IRES, RNA, Single molecule.

  • S. Calderari, M. Ria, C. Gérard, T. C. Nogueira, O. Villate, S. C. Collins, H. Neil, N. Gervasi, C. Hue, N. Suarez-Zamorano, C. Prado, M. Cnop, M. - T. Bihoreau, P. J. Kaisaki, J. - B. Cazier, C. Julier, M. Lathrop, M. Werner, D. L. Eizirik, and D. Gauguier, “Molecular genetics of the transcription factor GLIS3 identifies its dual function in beta cells and neurons”, Genomics, 2017.
    Abstract: The GLIS family zinc finger 3 isoform (GLIS3) is a risk gene for Type 1 and Type 2 diabetes, glaucoma and Alzheimer's disease endophenotype. We identified GLIS3 binding sites in insulin secreting cells (INS1) (FDR q<0.05; enrichment range 1.40-9.11 fold) sharing the motif wrGTTCCCArTAGs, which were enriched in genes involved in neuronal function and autophagy and in risk genes for metabolic and neuro-behavioural diseases. We confirmed experimentally Glis3-mediated regulation of the expression of genes involved in autophagy and neuron function in INS1 and neuronal PC12 cells. Naturally-occurring coding polymorphisms in Glis3 in the Goto-Kakizaki rat model of type 2 diabetes were associated with increased insulin production in vitro and in vivo, suggestive alteration of autophagy in PC12 and INS1 and abnormal neurogenesis in hippocampus neurons. Our results support biological pleiotropy of GLIS3 in pathologies affecting β-cells and neurons and underline the existence of trans‑nosology pathways in diabetes and its co-morbidities.
    Tags: Alzheimer's disease, ChIP sequencing, DBG, Diabetes mellitus, Goto-Kakizaki rat, GTR, Quantitative trait locus, Single nucleotide polymorphism.

  • K. Contrepois, C. Coudereau, B. A. Benayoun, N. Schuler, P. - F. Roux, O. Bischof, R. Courbeyrette, C. Carvalho, J. - Y. Thuret, Z. Ma, C. Derbois, M. - C. Nevers, H. Volland, C. E. Redon, W. M. Bonner, J. - F. Deleuze, C. Wiel, D. Bernard, M. P. Snyder, C. E. Rübe, R. Olaso, F. Fenaille, and C. Mann, “Histone variant H2A.J accumulates in senescent cells and promotes inflammatory gene expression”, Nature Communications, vol. 8, p. 14995, 2017.
    Abstract: The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases.
    Tags: DBG, SEN.

  • T. N. Dalia, S. H. Yoon, E. Galli, F. - X. Barre, C. M. Waters, and A. B. Dalia, “Enhancing multiplex genome editing by natural transformation (MuGENT) via inactivation of ssDNA exonucleases”, Nucleic Acids Research, 2017.
    Abstract: Recently, we described a method for multiplex genome editing by natural transformation (MuGENT). Mutant constructs for MuGENT require large arms of homology (>2000 bp) surrounding each genome edit, which necessitates laborious in vitro DNA splicing. In Vibrio cholerae, we uncover that this requirement is due to cytoplasmic ssDNA exonucleases, which inhibit natural transformation. In ssDNA exonuclease mutants, one arm of homology can be reduced to as little as 40 bp while still promoting integration of genome edits at rates of ∼50% without selection in cis. Consequently, editing constructs are generated in a single polymerase chain reaction where one homology arm is oligonucleotide encoded. To further enhance editing efficiencies, we also developed a strain for transient inactivation of the mismatch repair system. As a proof-of-concept, we used these advances to rapidly mutate 10 high-affinity binding sites for the nucleoid occlusion protein SlmA and generated a duodecuple mutant of 12 diguanylate cyclases in V. cholerae. Whole genome sequencing revealed little to no off-target mutations in these strains. Finally, we show that ssDNA exonucleases inhibit natural transformation in Acinetobacter baylyi. Thus, rational removal of ssDNA exonucleases may be broadly applicable for enhancing the efficacy and ease of MuGENT in diverse naturally transformable species.
    Tags: DBG, EMC2.

  • J. - F. Dallery, N. Lapalu, A. Zampounis, S. Pigné, I. Luyten, J. Amselem, A. H. J. Wittenberg, S. Zhou, M. V. de Queiroz, G. P. Robin, A. Auger, M. Hainaut, B. Henrissat, K. - T. Kim, Y. - H. Lee, O. Lespinet, D. C. Schwartz, M. R. Thon, and R. J. O'Connell, “Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters”, BMC genomics, vol. 18, no. 1, p. 667, 2017.
    Abstract: BACKGROUND: The ascomycete fungus Colletotrichum higginsianum causes anthracnose disease of brassica crops and the model plant Arabidopsis thaliana. Previous versions of the genome sequence were highly fragmented, causing errors in the prediction of protein-coding genes and preventing the analysis of repetitive sequences and genome architecture. RESULTS: Here, we re-sequenced the genome using single-molecule real-time (SMRT) sequencing technology and, in combination with optical map data, this provided a gapless assembly of all twelve chromosomes except for the ribosomal DNA repeat cluster on chromosome 7. The more accurate gene annotation made possible by this new assembly revealed a large repertoire of secondary metabolism (SM) key genes (89) and putative biosynthetic pathways (77 SM gene clusters). The two mini-chromosomes differed from the ten core chromosomes in being repeat- and AT-rich and gene-poor but were significantly enriched with genes encoding putative secreted effector proteins. Transposable elements (TEs) were found to occupy 7% of the genome by length. Certain TE families showed a statistically significant association with effector genes and SM cluster genes and were transcriptionally active at particular stages of fungal development. All 24 subtelomeres were found to contain one of three highly-conserved repeat elements which, by providing sites for homologous recombination, were probably instrumental in four segmental duplications. CONCLUSION: The gapless genome of C. higginsianum provides access to repeat-rich regions that were previously poorly assembled, notably the mini-chromosomes and subtelomeres, and allowed prediction of the complete SM gene repertoire. It also provides insights into the potential role of TEs in gene and genome evolution and host adaptation in this asexual pathogen.
    Tags: accessory chromosomes, BIM, Colletotrichum higginsianum, DBG, Fungal genome, optical map, secondary metabolism genes, segmental duplication, SMRT sequencing, subtelomeres, transposable elements.

  • A. Dostálová, S. Rommelaere, M. Poidevin, and B. Lemaitre, “Thioester-containing proteins regulate the Toll pathway and play a role in Drosophila defence against microbial pathogens and parasitoid wasps”, BMC biology, vol. 15, no. 1, p. 79, 2017.
    Abstract: BACKGROUND: Members of the thioester-containing protein (TEP) family contribute to host defence in both insects and mammals. However, their role in the immune response of Drosophila is elusive. In this study, we address the role of TEPs in Drosophila immunity by generating a mutant fly line, referred to as TEPq (Δ) , lacking the four immune-inducible TEPs, TEP1, 2, 3 and 4. RESULTS: Survival analyses with TEPq (Δ) flies reveal the importance of these proteins in defence against entomopathogenic fungi, Gram-positive bacteria and parasitoid wasps. Our results confirm that TEPs are required for efficient phagocytosis of bacteria, notably for the two Gram-positive species tested, Staphylococcus aureus and Enterococcus faecalis. Furthermore, we show that TEPq (Δ) flies have reduced Toll pathway activation upon microbial infection, resulting in lower expression of antimicrobial peptide genes. Epistatic analyses suggest that TEPs function upstream or independently of the serine protease ModSP at an initial stage of Toll pathway activation. CONCLUSIONS: Collectively, our study brings new insights into the role of TEPs in insect immunity. It reveals that TEPs participate in both humoral and cellular arms of immune response in Drosophila. In particular, it shows the importance of TEPs in defence against Gram-positive bacteria and entomopathogenic fungi, notably by promoting Toll pathway activation.
    Tags: Beauveria, Complement, DBG, Drosophila, Entomopathogenic fungus, EQYY, innate immunity, Insect, Parasitoid wasp, Phagocytosis.

  • E. Dubois, N. Mathy, V. Régnier, J. Bischerour, C. Baudry, R. Trouslard, and M. Bétermier, “Multimerization properties of PiggyMac, a domesticated piggyBac transposase involved in programmed genome rearrangements”, Nucleic Acids Research, 2017.
    Abstract: During sexual processes, the ciliate Paramecium eliminates 25-30% of germline DNA from its somatic genome. DNA elimination includes excision of ∼45 000 short, single-copy internal eliminated sequences (IESs) and depends upon PiggyMac (Pgm), a domesticated piggyBac transposase that is essential for DNA cleavage at IES ends. Pgm carries a core transposase region with a putative catalytic domain containing three conserved aspartic acids, and a downstream cysteine-rich (CR) domain. A C-terminal extension of unknown function is predicted to adopt a coiled-coil (CC) structure. To address the role of the three domains, we designed an in vivo complementation assay by expressing wild-type or mutant Pgm-GFP fusions in cells depleted for their endogenous Pgm. The DDD triad and the CR domain are essential for Pgm activity and mutations in either domain have a dominant-negative effect in wild-type cells. A mutant lacking the CC domain is partially active in the presence of limiting Pgm amounts, but inactive when Pgm is completely absent, suggesting that presence of the mutant protein increases the overall number of active complexes. We conclude that IES excision involves multiple Pgm subunits, of which at least a fraction must contain the CC domain.
    Tags: DBG, DSMC, MICMAC.

  • S. Duigou and F. Boccard, “Long range chromosome organization in Escherichia coli: The position of the replication origin defines the non-structured regions and the Right and Left macrodomains”, PLoS genetics, vol. 13, no. 5, p. e1006758, 2017.
    Abstract: The Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell.
    Tags: DBG, OCB.

  • T. Eychenne, M. Werner, and J. Soutourina, “Toward understanding of the mechanisms of Mediator function in vivo: Focus on the preinitiation complex assembly”, Transcription, p. e1329000, 2017.
    Abstract: Mediator is a multisubunit complex conserved in eukaryotes that plays an essential coregulator role in RNA polymerase (Pol) II transcription. Despite intensive studies of the Mediator complex, the molecular mechanisms of its function in vivo remain to be fully defined. In this review, we will discuss the different aspects of Mediator function starting with its interactions with specific transcription factors, its recruitment to chromatin and how, as a coregulator, it contributes to the assembly of transcription machinery components within the preinitiation complex (PIC) in vivo and beyond the PIC formation.
    Tags: coactivator, DBG, eukaryotic transcription, functional genomics, Genetics, GTR, human, Mediator, preinitiation complex, RNA Polymerase II, Structural Biology, yeast Saccharomyces cerevisiae.

  • P. J. Fabre, M. Leleu, B. H. Mormann, L. Lopez-Delisle, D. Noordermeer, L. Beccari, and D. Duboule, “Large scale genomic reorganization of topological domains at the HoxD locus”, Genome Biology, vol. 18, no. 1, p. 149, 2017.
    Abstract: BACKGROUND: The transcriptional activation of HoxD genes during mammalian limb development involves dynamic interactions with two topologically associating domains (TADs) flanking the HoxD cluster. In particular, the activation of the most posterior HoxD genes in developing digits is controlled by regulatory elements located in the centromeric TAD (C-DOM) through long-range contacts. RESULTS: To assess the structure-function relationships underlying such interactions, we measured compaction levels and TAD discreteness using a combination of chromosome conformation capture (4C-seq) and DNA FISH. We assessed the robustness of the TAD architecture by using a series of genomic deletions and inversions that impact the integrity of this chromatin domain and that remodel long-range contacts. We report multi-partite associations between HoxD genes and up to three enhancers. We find that the loss of native chromatin topology leads to the remodeling of TAD structure following distinct parameters. CONCLUSIONS: Our results reveal that the recomposition of TAD architectures after large genomic re-arrangements is dependent on a boundary-selection mechanism in which CTCF mediates the gating of long-range contacts in combination with genomic distance and sequence specificity. Accordingly, the building of a recomposed TAD at this locus depends on distinct functional and constitutive parameters.
    Tags: CHRODY, Chromatin organization, CTCF, DBG, Enhancer, Gene regulation, Hox, Limb development, Regulatory landscape, TAD, Topologically associating domains.


  • B. Felden and P. Bouloc, “Regulatory RNAs in bacteria: From identification to function”, Methods, vol. 117, p. 1-2, 2017.


  • S. Fieulaine, R. Alves de Sousa, L. Maigre, K. Hamiche, M. Alimi, J. - M. Bolla, A. Taleb, A. Denis, J. - M. Pagès, I. Artaud, T. Meinnel, and C. Giglione, “Corrigendum: A unique peptide deformylase platform to rationally design and challenge novel active compounds”, Scientific Reports, vol. 7, p. 39365, Jan. 2017.


  • E. Galli, C. Midonet, E. Paly, and F. - X. Barre, “Fast growth conditions uncouple the final stages of chromosome segregation and cell division in Escherichia coli”, PLOS Genetics, vol. 13, no. 3, p. e1006702, Mar. 2017.


  • E. Galli, E. Paly, and F. - X. Barre, “Late assembly of the Vibrio cholerae cell division machinery postpones septation to the last 10% of the cell cycle”, Scientific Reports, vol. 7, p. 44505, Mar. 2017.

  • A. Glatigny, P. Gambette, A. Bourand-Plantefol, G. Dujardin, and M. - H. Mucchielli-Giorgi, “Development of an in silico method for the identification of subcomplexes involved in the biogenesis of multiprotein complexes in Saccharomyces cerevisiae”, BMC systems biology, vol. 11, no. 1, p. 67, 2017.
    Abstract: BACKGROUND: Large sets of protein-protein interaction data coming either from biological experiments or predictive methods are available and can be combined to construct networks from which information about various cell processes can be extracted. We have developed an in silico approach based on these information to model the biogenesis of multiprotein complexes in the yeast Saccharomyces cerevisiae. RESULTS: Firstly, we have built three protein interaction networks by collecting the protein-protein interactions, which involved the subunits of three complexes, from different databases. The protein-protein interactions come from different kinds of biological experiments or are predicted. We have chosen the elongator and the mediator head complexes that are soluble and exhibit an architecture with subcomplexes that could be functional modules, and the mitochondrial bc 1 complex, which is an integral membrane complex and for which a late assembly subcomplex has been described. Secondly, by applying a clustering strategy to these networks, we were able to identify subcomplexes involved in the biogenesis of the complexes as well as the proteins interacting with each subcomplex. Thirdly, in order to validate our in silico results for the cytochrome bc1 complex we have analysed the physical interactions existing between three subunits by performing immunoprecipitation experiments in several genetic context. CONCLUSIONS: For the two soluble complexes (the elongator and mediator head), our model shows a strong clustering of subunits that belong to a known subcomplex or module. For the membrane bc 1 complex, our approach has suggested new interactions between subunits in the early steps of the assembly pathway that were experimentally confirmed. Scripts can be downloaded from the site: http://bim.igmors.u-psud.fr/isips .
    Tags: BIM, BIOCELL, BIOMIT, Complex assembly, DBG, Graph clustering, PPI network, Protein complex, Protein-protein interactions, Subcomplex.

  • J. Gruchota, C. Denby Wilkes, O. Arnaiz, L. Sperling, and J. K. Nowak, “A meiosis-specific Spt5 homolog involved in non-coding transcription”, Nucleic Acids Research, 2017.
    Abstract: Spt5 is a conserved and essential transcriptional regulator that binds directly to RNA polymerase and is involved in transcription elongation, polymerase pausing and various co-transcriptional processes. To investigate the role of Spt5 in non-coding transcription, we used the unicellular model Paramecium tetraurelia In this ciliate, development is controlled by epigenetic mechanisms that use different classes of non-coding RNAs to target DNA elimination. We identified two SPT5 genes. One (STP5v) is involved in vegetative growth, while the other (SPT5m) is essential for sexual reproduction. We focused our study on SPT5m, expressed at meiosis and associated with germline nuclei during sexual processes. Upon Spt5m depletion, we observed absence of scnRNAs, piRNA-like 25 nt small RNAs produced at meiosis. The scnRNAs are a temporal copy of the germline genome and play a key role in programming DNA elimination. Moreover, Spt5m depletion abolishes elimination of all germline-limited sequences, including sequences whose excision was previously shown to be scnRNA-independent. This suggests that in addition to scnRNA production, Spt5 is involved in setting some as yet uncharacterized epigenetic information at meiosis. Our study establishes that Spt5m is crucial for developmental genome rearrangements and necessary for scnRNA production.
    Tags: ANGE, DBG.

  • R. Grzela, J. Nusbaum, S. Fieulaine, F. Lavecchia, W. V. Bienvenut, C. Dian, T. Meinnel, and C. Giglione, “The C-terminal residue of phage Vp16 PDF, the smallest peptide deformylase, acts as an offset element locking the active conformation”, Scientific Reports, vol. 7, no. 1, p. 11041, 2017.
    Abstract: Prokaryotic proteins must be deformylated before the removal of their first methionine. Peptide deformylase (PDF) is indispensable and guarantees this mechanism. Recent metagenomics studies revealed new idiosyncratic PDF forms as the most abundant family of viral sequences. Little is known regarding these viral PDFs, including the capacity of the corresponding encoded proteins to ensure deformylase activity. We provide here the first evidence that viral PDFs, including the shortest PDF identified to date, Vp16 PDF, display deformylase activity in vivo, despite the absence of the key ribosome-interacting C-terminal region. Moreover, characterization of phage Vp16 PDF underscores unexpected structural and molecular features with the C-terminal Isoleucine residue significantly contributing to deformylase activity both in vitro and in vivo. This residue fully compensates for the absence of the usual long C-domain. Taken together, these data elucidate an unexpected mechanism of enzyme natural evolution and adaptation within viral sequences.
    Tags: B3S, DBG, IMAPP, PROMTI.

  • F. Guérin, O. Arnaiz, N. Boggetto, C. Denby Wilkes, E. Meyer, L. Sperling, and S. Duharcourt, “Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements”, BMC genomics, vol. 18, no. 1, p. 327, 2017.
    Abstract: BACKGROUND: DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences. RESULTS: We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome. CONCLUSIONS: We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.
    Tags: ANGE, DBG, Flow Cytometry, High throughput sequencing, ITm DNA transposons, Non-LTR retrotransposons, Programmed DNA elimination.

  • H. Lalucque, F. Malagnac, K. Green, V. Gautier, P. Grognet, L. Chan Ho Tong, B. Scott, and P. Silar, “IDC2 and IDC3, two genes involved in cell non-autonomous signaling of fruiting body development in the model fungus Podospora anserina”, Developmental Biology, vol. 421, no. 2, p. 126-138, 2017.
    Abstract: Filamentous ascomycetes produce complex multicellular structures during sexual reproduction. Little is known about the genetic pathways enabling the construction of such structures. Here, with a combination of classical and reverse genetic methods, as well as genetic mosaic and graft analyses, we identify and provide evidence for key roles for two genes during the formation of perithecia, the sexual fruiting bodies, of the filamentous fungus Podospora anserina. Data indicate that the proteins coded by these two genes function cell-non-autonomously and that their activity depends upon conserved cysteines, making them good candidate for being involved in the transmission of a reactive oxygen species (ROS) signal generated by the PaNox1 NADPH oxidase inside the maturing fruiting body towards the PaMpk1 MAP kinase, which is located inside the underlying mycelium, in which nutrients are stored. These data provide important new insights to our understanding of how fungi build multicellular structures.
    Tags: DBG, Developmental mutants, DSMC, Fungal development, Multicellular fruiting bodies, Perithecium, Podospora anserina.

  • J. Lehmann, “Induced fit of the peptidyl-transferase center of the ribosome and conformational freedom of the esterified amino acids”, RNA (New York, N.Y.), vol. 23, no. 2, p. 229-239, 2017.
    Abstract: The catalytic site of most enzymes can efficiently handle only one substrate. In contrast, the ribosome is capable of polymerizing at a similar rate at least 20 different kinds of amino acids from aminoacyl-tRNA carriers while using just one catalytic site, the peptidyl-transferase center (PTC). An induced-fit mechanism has been uncovered in the PTC, but a possible connection between this mechanism and the uniform handling of the substrates has not been investigated. We present an analysis of published ribosome structures supporting the hypothesis that the induced fit eliminates unreactive rotamers predominantly populated for some A-site aminoacyl esters before induction. We show that this hypothesis is fully consistent with the wealth of kinetic data obtained with these substrates. Our analysis reveals that induction constrains the amino acids into a reactive conformation in a side-chain independent manner. It allows us to highlight the rationale of the PTC structural organization, which confers to the ribosome the very unusual ability to handle large as well as small substrates.
    Tags: aminoacyl-tRNA, DBG, EF-P, induced fit, peptidyl-transferase center, Ribosome, SSFA.

  • S. Li, E. Porcel, H. Remita, S. Marco, M. Réfrégiers, M. Dutertre, F. Confalonieri, and S. Lacombe, “Platinum nanoparticles: an exquisite tool to overcome radioresistance”, Cancer Nanotechnology, vol. 8, no. 1, p. 4, 2017.
    Abstract: BACKGROUD: Small metallic nanoparticles are proposed as potential nanodrugs to optimize the performances of radiotherapy. This strategy, based on the enrichment of tumours with nanoparticles to amplify radiation effects in the tumour, aims at increasing the cytopathic effect in tumours while healthy tissue is preserved, an important challenge in radiotherapy. Another major cause of radiotherapy failure is the radioresistance of certain cancers. Surprisingly, the use of nanoparticles to overcome radioresistance has not, to the best of our knowledge, been extensively investigated. The mechanisms of radioresistance have been extensively studied using Deinococcus radiodurans, the most radioresistant organism ever reported, as a model. METHODS: In this work, we investigated the impact of ultra-small platinum nanoparticles (1.7 nm) on this organism, including uptake, toxicity, and effects on radiation responses. RESULTS: We showed that the nanoparticles penetrate D. radiodurans cells, despite the 150 nm cell wall thickness with a minimal inhibition concentration on the order of 4.8 mg L(-1). We also found that the nanoparticles amplify gamma ray radiation effects by >40%. CONCLUSIONS: Finally, this study demonstrates the capacity of metallic nanoparticles to amplify radiation in radioresistant organisms, thus opening the perspective to use nanoparticles not only to improve tumour targeting but also to overcome radioresistance.
    Tags: DBG, Deinococcus radiodurans, Metallic nanoparticles, Radio-enhancement, Radioresistance, Radiosensitization, RBA.


  • B. Michel and S. J. Sandler, “Replication Restart in Bacteria”, Journal of Bacteriology, p. JB.00102-17, Mar. 2017.

  • B. Michel and A. K. Sinha, “The inactivation of rfaP, rarA or sspA gene improves the viability of the Escherichia coli DNA polymerase III holD mutant”, Molecular Microbiology, 2017.
    Abstract: The Escherichia coli holD mutant is poorly viable because the stability of holoenzyme polymerase III (Pol III HE) on DNA is compromised. Consequently, the SOS response is induced and the SOS polymerases DinB and Pol II further hinder replication. Mutations that restore the holD mutant viability belong to two classes, those that stabilize Pol III on DNA and those that prevent the deleterious effects of DinB over-production. We identified a dnaX mutation and the inactivation of rfaP and sspA genes as belonging to the first class of holD mutant suppressors. dnaX encodes a Pol III clamp loader subunit that interacts with HolD. rfaP encodes a lipopolysaccharide kinase that acts in outer membrane biogenesis. Its inactivation improves the holD mutant growth in part by affecting potassium import, previously proposed to stabilize Pol III HE on DNA by increasing electrostatic interactions. sspA encodes a global transcriptional regulator and growth of the holD mutant in its absence suggests that SspA controls genes that affect protein-DNA interactions. The inactivation of rarA belongs to the second class of suppressor mutations. rarA inactivation has a weak effect but is additive with other suppressor mutations. Our results suggest that RarA facilitates DinB binding to abandoned forks.
    Tags: DBG, STABAC.

  • M. Mirande, “The Aminoacyl-tRNA Synthetase Complex”, Sub-Cellular Biochemistry, vol. 83, p. 505-522, 2017.
    Abstract: Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that specifically aminoacylate one tRNA molecule by the cognate amino acid. They are a family of twenty enzymes, one for each amino acid. By coupling an amino acid to a specific RNA triplet, the anticodon, they are responsible for interpretation of the genetic code. In addition to this translational, canonical role, several aminoacyl-tRNA synthetases also fulfill nontranslational, moonlighting functions. In mammals, nine synthetases, those specific for amino acids Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met and Pro, associate into a multi-aminoacyl-tRNA synthetase complex, an association which is believed to play a key role in the cellular organization of translation, but also in the regulation of the translational and nontranslational functions of these enzymes. Because the balance between their alternative functions rests on the assembly and disassembly of this supramolecular entity, it is essential to get precise insight into the structural organization of this complex. The high-resolution 3D-structure of the native particle, with a molecular weight of about 1.5 MDa, is not yet known. Low-resolution structures of the multi-aminoacyl-tRNA synthetase complex, as determined by cryo-EM or SAXS, have been reported. High-resolution data have been reported for individual enzymes of the complex, or for small subcomplexes. This review aims to present a critical view of our present knowledge of the aminoacyl-tRNA synthetase complex in 3D. These preliminary data shed some light on the mechanisms responsible for the balance between the translational and nontranslational functions of some of its components.
    Tags: Aminoacyl-tRNA synthetase (AARS), Core synthetases, Crystal Structure, DBG, MARS, MSC assembly, Multi-aminacyl-tRNA synthetase complex (MSC).

  • M. Nakashima, R. Yamagami, C. Tomikawa, Y. Ochi, T. Moriya, H. Asahara, D. Fourmy, S. Yoshizawa, T. Oshima, and H. Hori, “Long and branched polyamines are required for maintenance of the ribosome, tRNA(His) and tRNA(Tyr) in Thermus thermophilus cells at high temperatures”, Genes to Cells: Devoted to Molecular & Cellular Mechanisms, 2017.
    Abstract: Thermus thermophilus is an extremely thermophilic eubacterium that produces various polyamines. Aminopropylagmatine ureohydrolase (SpeB) and SAM decarboxylase-like protein 1 (SpeD1) are involved in the biosynthesis of spermidine from arginine. Because long and branched polyamines in T. thermophilus are synthesized from spermidine, the speB and speD1 gene-deleted strains (ΔspeB and ΔspeD1, respectively) cannot synthesize long and branched polyamines. Although neither strain grew at high temperatures (>75°C) in minimal medium, both strains survived at 80°C when they were cultured at 70°C until the mid-log phase and then shifted to 80°C. We therefore prepared the ΔspeB and ΔspeD1 cells using this culture method. Microscopic analysis showed that both strains can survive for 10 h after the temperature shift. Although the modification levels of 2'-O-methylguanosine at position 18, N(7) -methylguanosine at position 46, 5-methyluridine at position 54 and N(1) -methyladenosine at position 58 in the class I tRNA from both strains were normal, amounts of tRNA(Tyr) , tRNA(His) , rRNAs and 70S ribosomes were decreased after the temperature shift. Furthermore, in vivo protein synthesis in both strains was completely lost 10 h after the temperature shift. Thus, long and branched polyamines are required for at least the maintenance of 70S ribosome and some tRNA species at high temperatures.
    Tags: DBG, RNASTR.

  • T. Plénat, S. Yoshizawa, and D. Fourmy, “DNA-Guided Delivery of Single Molecules into Zero-Mode Waveguides”, ACS applied materials & interfaces, 2017.
    Abstract: Zero-mode waveguides (ZMWs) are powerful analytical tools corresponding to optical nanostructures fabricated in a thin metallic film capable of confining an excitation volume to the range of attoliters. This small volume of confinement allows single-molecule fluorescence experiments to be performed at physiologically relevant concentrations of fluorescently labelled biomolecules. Exactly one molecule to be studied must be attached at the floor of the ZMW for signal detection and analysis; however, the massive parallelism of these nanoarrays suffers from a Poissonian-limited distribution of these biomolecules. To date, there is no method available that provides full single molecule occupancy of massively arrayed ZMWs. Here we report the performance of a DNA-guided method that uses steric exclusion properties of large DNA molecules to bias the Poissonian-limited delivery of single molecules. Non-Poissonian statistics were obtained with DNA molecules that contain a free-biotinylated extremity for efficient binding to the floor of the ZMW, which resulted in a decrease of accessibility for a second molecule. Both random coil and condensed DNA conformations drove non-Poissonian single-molecule delivery into ZMWs arrays. The results suggest that an optimal balance between rigidity and flexibility of the macromolecule is critical for favourable accessibility and single occupancy. The optimized method provides means for full exploitation of these massively parallelized analytical tools.
    Tags: DBG, RNASTR.

  • M. Poidevin, E. Galli, Y. Yamaichi, and F. - X. Barre, “WGADseq: Whole Genome Affinity Determination of Protein-DNA Binding Sites”, Methods in Molecular Biology (Clifton, N.J.), vol. 1624, p. 53-60, 2017.
    Abstract: We present a method through which one may monitor the relative binding affinity of a given protein to DNA motifs on the scale of a whole genome. Briefly, the protein of interest is incubated with fragmented genomic DNA and then affixed to a column. Washes with buffers containing low salt concentrations will remove nonbound DNA fragments, while stepwise washes with increasing salt concentrations will elute more specifically bound fragments. Massive sequencing is used to identify eluted DNA fragments and map them on the genome, which permits us to classify the different binding sites according to their affinity and determine corresponding consensus motifs (if any).
    Tags: DBG, EMC2, EQYY, Genomics, High-throughput sequencing, Next-generation sequencing (NGS), Site-specific DNA binding protein.

  • R. I. Ponce-Toledo, P. Deschamps, P. López-García, Y. Zivanovic, K. Benzerara, and D. Moreira, “An Early-Branching Freshwater Cyanobacterium at the Origin of Plastids”, Current biology: CB, vol. 27, no. 3, p. 386-391, 2017.
    Abstract: Photosynthesis evolved in eukaryotes by the endosymbiosis of a cyanobacterium, the future plastid, within a heterotrophic host. This primary endosymbiosis occurred in the ancestor of Archaeplastida, a eukaryotic supergroup that includes glaucophytes, red algae, green algae, and land plants [1-4]. However, although the endosymbiotic origin of plastids from a single cyanobacterial ancestor is firmly established, the nature of that ancestor remains controversial: plastids have been proposed to derive from either early- or late-branching cyanobacterial lineages [5-11]. To solve this issue, we carried out phylogenomic and supernetwork analyses of the most comprehensive dataset analyzed so far including plastid-encoded proteins and nucleus-encoded proteins of plastid origin resulting from endosymbiotic gene transfer (EGT) of primary photosynthetic eukaryotes, as well as wide-ranging genome data from cyanobacteria, including novel lineages. Our analyses strongly support that plastids evolved from deep-branching cyanobacteria and that the present-day closest cultured relative of primary plastids is Gloeomargarita lithophora. This species belongs to a recently discovered cyanobacterial lineage widespread in freshwater microbialites and microbial mats [12, 13]. The ecological distribution of this lineage sheds new light on the environmental conditions where the emergence of photosynthetic eukaryotes occurred, most likely in a terrestrial-freshwater setting. The fact that glaucophytes, the first archaeplastid lineage to diverge, are exclusively found in freshwater ecosystems reinforces this hypothesis. Therefore, not only did plastids emerge early within cyanobacteria, but the first photosynthetic eukaryotes most likely evolved in terrestrial-freshwater settings, not in oceans as commonly thought.
    Tags: chloroplasts, Cyanobacteria, DBG, evolution, molecular phylogeny, phylogenomics, Plastids, RBA.


  • W. V. Bienvenut, C. Giglione, and T. Meinnel, “SILProNAQ: A Convenient Approach for Proteome-Wide Analysis of Protein N-Termini and N-Terminal Acetylation Quantitation”, in Protein Terminal Profiling, vol. 1574, O. Schilling, Eds. New York, NY: Springer New York, 2017, p. 17-34.

  • L. Shi, K. France, O. Arnaiz, and J. Cohen, “The Ciliary Protein IFT57 in the Macronucleus of Paramecium”, The Journal of Eukaryotic Microbiology, 2017.
    Abstract: The intraflagellar transport IFT57 protein is essential for ciliary growth and maintenance. Also known as HIPPI, human IFT57 can be translocated to the nucleus via a molecular partner of the Huntingtin, Hip1, inducing gene expression changes. In Paramecium tetraurelia, we identified four IFT57 genes forming two subfamilies IFT57A/B and IFT57C/D arising from whole genome duplications. The depletion of proteins of the two subfamilies induced ciliary defects and IFT57A and IFT57C localized in basal bodies and cilia. We observed that IFT57A, but not IFT57C, is also present in the macronucleus and able to traffic toward the developing anlage during autogamy. Analysis of chimeric IFT57A-IFT57C-GFP-tagged proteins allowed us to identify a region of IFT57A necessary for nuclear localization. We studied the localization of the unique IFT57 protein of Paramecium caudatum, a species, which diverged from Paramecium tetraurelia before the whole genome duplications. The Paramecium caudatum IFT57C protein was excluded from the nucleus. We also analyzed whether the overexpression of IFT57A in Paramecium could affect gene transcription as the human protein does in HeLa cells. The expression of some genes was indeed affected by overexpression of IFT57A, but the set of affected genes poorly overlaps the set of genes affected in human cells. This article is protected by copyright. All rights reserved.
    Tags: ANGE, BIOCELL, BIOCIL, cilia, DBG, IFT57 /HIPPI, intraflagellar transport (IFT), Macronucleus, Paramecium.

  • F. Simonetti, T. Candelli, S. Leon, D. Libri, and M. Rougemaille, “Ubiquitination-dependent control of sexual differentiation in fission yeast”, eLife, vol. 6, 2017.
    Abstract: In fission yeast, meiosis-specific transcripts are selectively eliminated during vegetative growth by the combined action of the YTH-family RNA-binding protein Mmi1 and the nuclear exosome. Upon nutritional starvation, the master regulator of meiosis Mei2 inactivates Mmi1, thereby allowing expression of the meiotic program. Here, we show that the E3 ubiquitin ligase subunit Not4/Mot2 of the evolutionarily conserved Ccr4-Not complex, which associates with Mmi1, promotes suppression of meiotic transcripts expression in mitotic cells. Our analyses suggest that Mot2 directs ubiquitination of Mei2 to preserve the activity of Mmi1 during vegetative growth. Importantly, Mot2 is not involved in the constitutive pathway of Mei2 turnover, but rather plays a regulatory role to limit its accumulation or inhibit its function. We propose that Mmi1 recruits the Ccr4-Not complex to counteract its own inhibitor Mei2, thereby locking the system in a stable state that ensures the repression of the meiotic program by Mmi1.
    Tags: Chromosomes, DBG, genes, GEXDIF, S. pombe.

  • A. Thiébaut, T. Delaveau, M. Benchouaia, J. Boeri, M. Garcia, G. Lelandais, and F. Devaux, “The CCAAT-Binding Complex Controls Respiratory Gene Expression and Iron Homeostasis in Candida Glabrata”, Scientific Reports, vol. 7, no. 1, p. 3531, 2017.
    Abstract: The CCAAT-binding complex (CBC) is a heterotrimeric transcription factor which is widely conserved in eukaryotes. In the model yeast S. cerevisiae, CBC positively controls the expression of respiratory pathway genes. This role involves interactions with the regulatory subunit Hap4. In many pathogenic fungi, CBC interacts with the HapX regulatory subunit to control iron homeostasis. HapX is a bZIP protein which only shares with Hap4 the Hap4Like domain (Hap4L) required for its interaction with CBC. Here, we show that CBC has a dual role in the pathogenic yeast C. glabrata. It is required, along with Hap4, for the constitutive expression of respiratory genes and it is also essential for the iron stress response, which is mediated by the Yap5 bZIP transcription factor. Interestingly, Yap5 contains a vestigial Hap4L domain. The mutagenesis of this domain severely reduced Yap5 binding to its targets and compromised its interaction with Hap5. Hence, Yap5, like HapX in other species, acts as a CBC regulatory subunit in the regulation of iron stress response. This work reveals new aspects of iron homeostasis in C. glabrata and of the evolution of the role of CBC and Hap4L-bZIP proteins in this process.
    Tags: BIM, DBG.

  • E. Thierion, J. Le Men, S. Collombet, C. Hernandez, F. Coulpier, P. Torbey, M. Thomas-Chollier, D. Noordermeer, P. Charnay, and P. Gilardi-Hebenstreit, “Krox20 hindbrain regulation incorporates multiple modes of cooperation between cis-acting elements”, PLoS genetics, vol. 13, no. 7, p. e1006903, 2017.
    Abstract: Developmental genes can harbour multiple transcriptional enhancers that act simultaneously or in succession to achieve robust and precise spatiotemporal expression. However, the mechanisms underlying cooperation between cis-acting elements are poorly documented, notably in vertebrates. The mouse gene Krox20 encodes a transcription factor required for the specification of two segments (rhombomeres) of the developing hindbrain. In rhombomere 3, Krox20 is subject to direct positive feedback governed by an autoregulatory enhancer, element A. In contrast, a second enhancer, element C, distant by 70 kb, is active from the initiation of transcription independent of the presence of the KROX20 protein. Here, using both enhancer knock-outs and investigations of chromatin organisation, we show that element C possesses a dual activity: besides its classical enhancer function, it is also permanently required in cis to potentiate the autoregulatory activity of element A, by increasing its chromatin accessibility. This work uncovers a novel, asymmetrical, long-range mode of cooperation between cis-acting elements that might be essential to avoid promiscuous activation of positive autoregulatory elements.
    Tags: Animals, Body Patterning, CHRODY, Chromatin, DBG, Early Growth Response Protein 1, Enhancer Elements, Genetic, Gene Expression Regulation, Developmental, Mice, Knockout, Mutation, Regulatory Elements, Transcriptional, Rhombencephalon, Sequence Homology, Nucleic Acid.


  • C. Voisset, M. Blondel, G. W. Jones, G. Friocourt, G. Stahl, S. Chédin, V. Beringue, and R. Gillet, “The double life of the ribosome: when its protein folding activity supports prion propagation”, Prion, p. 00-00, Mar. 2017.

  • S. Wang, T. Hassold, P. Hunt, M. A. White, D. Zickler, N. Kleckner, and L. Zhang, “Inefficient Crossover Maturation Underlies Elevated Aneuploidy in Human Female Meiosis”, Cell, vol. 168, no. 6, p. 977-989.e17, 2017.
    Abstract: Meiosis is the cellular program that underlies gamete formation. For this program, crossovers between homologous chromosomes play an essential mechanical role to ensure regular segregation. We present a detailed study of crossover formation in human male and female meiosis, enabled by modeling analysis. Results suggest that recombination in the two sexes proceeds analogously and efficiently through most stages. However, specifically in female (but not male), ∼25% of the intermediates that should mature into crossover products actually fail to do so. Further, this "female-specific crossover maturation inefficiency" is inferred to make major contributions to the high level of chromosome mis-segregation and resultant aneuploidy that uniquely afflicts human female oocytes (e.g., giving Down syndrome). Additionally, crossover levels on different chromosomes in the same nucleus tend to co-vary, an effect attributable to global per-nucleus modulation of chromatin loop size. Maturation inefficiency could potentially reflect an evolutionary advantage of increased aneuploidy for human females.
    Tags: DBG, DSMC.

  • N. Xie, G. Ruprich-Robert, F. Chapeland-Leclerc, E. Coppin, H. Lalucque, S. Brun, R. Debuchy, and P. Silar, “Inositol-phosphate signaling as mediator for growth and sexual reproduction in Podospora anserina”, Developmental Biology, 2017.
    Abstract: The molecular pathways involved in the development of multicellular fruiting bodies in fungi are still not well known. Especially, the interplay between the mycelium, the female tissues and the zygotic tissues of the fruiting bodies is poorly documented. Here, we describe PM154, a new strain of the model ascomycetes Podospora anserina able to mate with itself and that enabled the easy recovery of new mutants affected in fruiting body development. By complete genome sequencing of spod1, one of the new mutants, we identified an inositol phosphate polykinase gene as essential, especially for fruiting body development. A factor present in the wild type and diffusible in mutant hyphae was able to induce the development of the maternal tissues of the fruiting body in spod1, but failed to promote complete development of the zygotic ones. Addition of myo-inositol in the growth medium was able to increase the number of developing fruiting bodies in the wild type, but not in spod1. Overall, the data indicated that inositol and inositol polyphosphates were involved in promoting fruiting body maturation, but also in regulating the number of fruiting bodies that developed after fertilization. The same effect of inositol was seen in two other fungi, Sordaria macrospora and Chaetomium globosum. Key role of the inositol polyphosphate pathway during fruiting body maturation appears thus conserved during the evolution of Sordariales fungi.
    Tags: DBG, Developmental mutants, DSMC, Fungal development, Inositol, Inositol kinase, Multicellular fruiting bodies, Perithecium, Podospora anserina.

  • Y. Yamaichi and T. Dörr, “Transposon Insertion Site Sequencing for Synthetic Lethal Screening”, Methods in Molecular Biology (Clifton, N.J.), vol. 1624, p. 39-49, 2017.
    Abstract: Transposon insertion site sequencing (TIS) permits genome-wide, quantitative fitness assessment of individual genomic loci. In addition to the identification of essential genes in given growth conditions, TIS enables the elucidation of genetic networks such as synthetic lethal or suppressor gene combinations. Therefore, TIS becomes an exceptionally powerful tool for the high-throughput determination of genotype-phenotype relationships in bacteria. Here, we describe a protocol for the generation of high-density transposon insertion libraries and subsequent preparation of DNA samples for Illumina sequencing using the Gram-negative bacterium Vibrio cholerae as an example.
    Tags: DBG, EQYY, Fitness, Genomics, High-throughput sequencing, Mariner transposon, TIS, TnSeq, Transposon mutagenesis.

2016


  • E. Allemand, M. P. Myers, J. Garcia-Bernardo, A. Harel-Bellan, A. R. Krainer, and C. Muchardt, “A Broad Set of Chromatin Factors Influences Splicing”, PLoS genetics, vol. 12, no. 9, p. e1006318, 2016.
    Abstract: Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing.
    Tags: DBG, LEC.


  • S. Bakari, M. Lembrouk, L. Sourd, F. Ousalem, F. André, S. Orlowski, M. Delaforge, and A. Frelet-Barrand, “Lactococcus lactis is an Efficient Expression System for Mammalian Membrane Proteins Involved in Liver Detoxification, CYP3A4, and MGST1”, Molecular Biotechnology, vol. 58, no. 4, p. 299-310, 2016.

  • R. Balbontín, N. Villagra, M. Pardos de la Gándara, G. Mora, N. Figueroa-Bossi, and L. Bossi, “Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues”, Molecular Microbiology, vol. 100, no. 1, p. 139-155, 2016.
    Abstract: The iroN gene of Salmonella enterica and uropathogenic Escherichia coli encodes the outer membrane receptor of Fe(3+) -bound salmochelin, a siderophore tailored to evade capture by the host's immune system. The iroN gene is under negative control of the Fur repressor and transcribed under iron limiting conditions. We show here that transcriptional de-repression is not sufficient to allow iroN expression, as this also requires activation by either of two partially homologous small RNAs (sRNAs), RyhB1 and RyhB2. The two sRNAs target the same sequence segment approximately in the middle of the 94-nucleotide 5' untranslated region (UTR) of iroN mRNA. Several lines of evidence suggest that base pair interaction stimulates iroN mRNA translation. Activation does not result from the disruption of a secondary structure masking the ribosome binding site; rather it involves sequences at the 5' end of iroN 5' UTR. In vitro 'toeprint' assays revealed that this upstream site binds the 30S ribosomal subunit provided that RyhB1 is paired with the mRNA. Altogether, our data suggest that RyhB1, and to lesser extent RyhB2, activate iroN mRNA translation by promoting entry of the ribosome at an upstream 'standby' site. These findings add yet an additional nuance to the polychromatic landscape of sRNA-mediated regulation.
    Tags: 5' Untranslated Regions, Bacterial Outer Membrane Proteins, Bacterial Proteins, Base Sequence, Binding Sites, Codon, Initiator, Conserved Sequence, DBG, Gene Expression Regulation, Bacterial, Nucleic Acid Conformation, Nucleotide Motifs, Protein Binding, Receptors, Cell Surface, RGSP, Ribosomes, RNA Stability, RNA, Bacterial, RNA, Messenger, RNA-Binding Proteins.

  • E. Barbier, A. Lagorce, A. Hachemi, M. Dutertre, A. Gorlas, L. Morand, C. Saint-Pierre, J. - L. Ravanat, T. Douki, J. Armengaud, D. Gasparutto, F. Confalonieri, and J. Breton, “Oxidative DNA Damage and Repair in the Radioresistant Archaeon Thermococcus gammatolerans”, Chemical Research in Toxicology, vol. 29, no. 11, p. 1796-1809, 2016.
    Abstract: The hyperthermophilic archaeon Thermococcus gammatolerans can resist huge doses of γ-irradiation, up to 5.0 kGy, without loss of viability. The potential to withstand such harsh conditions is probably due to complementary passive and active mechanisms, including repair of damaged chromosomes. In this work, we documented the formation and repair of oxidative DNA lesions in T. gammatolerans. The basal level of the oxidized nucleoside, 8-oxo-2'-deoxyguanosine (8-oxo-dGuo), was established at 9.2 (± 0.9) 8-oxo-dGuo per 10(6) nucleosides, a higher level than those usually measured in eukaryotic cells or bacteria. A significant increase in oxidative damage, i.e., up to 24.2 (± 8.0) 8-oxo-dGuo/10(6) nucleosides, was measured for T. gammatolerans exposed to a 5.0 kGy dose of γ-rays. Surprisingly, the yield of radiation-induced modifications was lower than those previously observed for human cells exposed to doses corresponding to a few grays. One hour after irradiation, 8-oxo-dGuo levels were significantly reduced, indicating an efficient repair. Two putative base excision repair (BER) enzymes, TGAM_1277 and TGAM_1653, were demonstrated both by proteomics and transcriptomics to be present in the cells without exposure to ionizing radiation. Their transcripts were moderately upregulated after gamma irradiation. After heterologous production and purification of these enzymes, biochemical assays based on electrophoresis and MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry indicated that both have a β-elimination cleavage activity. TGAM_1653 repairs 8-oxo-dGuo, whereas TGAM_1277 is also able to remove lesions affecting pyrimidines (1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd)). This work showed that in normal growth conditions or in the presence of a strong oxidative stress, T. gammatolerans has the potential to rapidly reduce the extent of DNA oxidation, with at least these two BER enzymes as bodyguards with distinct substrate ranges.
    Tags: DBG, RBA.

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