51. A Novel Toxoplasma gondii Nuclear Factor TgNF3 Is a Dynamic Chromatin-Associated Component, Modulator of Nucleolar Architecture and Parasite Virulence
- Author
-
Agnès Hovasse, Christine Schaeffer-Reiss, Alain Van Dorsselaer, Thomas Mouveaux, Alejandro Olguin-Lamas, Stanislas Tomavo, Edwige Madec, Christian Slomianny, Elisabeth Werkmeister, Stéphane Delhaye, Isabelle Callebaut, Centre d’Infection et d’Immunité de Lille - INSERM U 1019 - UMR 9017 - UMR 8204 (CIIL), Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Institut de minéralogie et de physique des milieux condensés (IMPMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-IPG PARIS-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Rôle des canaux ioniques membranaires et du calcium intracellulaire dans la physiopathologie de la prostate, Université de Lille, Sciences et Technologies-Institut National de la Santé et de la Recherche Médicale (INSERM), This research was funded by the Centre National de la Recherche Scientifique (CNRS), Institut National de la Sante et de la Recherche Medicale(INSERM), Institut Pasteur de Lille (IPL). The funders had no role in study design, data collection and analysis, decision to publish, or preparationof the manuscript., We would like to thank Dewailly E and Mortuaire M for excellent technical assistance, especially in the earlier phase of this study. We acknowledge Drs Harb O and Roos DS (University of Pennsylvania) for providing the nucleotide sequence of T. gondii rDNA 18S. We are indebted to Drs Hakimi A and Leprince D for providing nucleosome and antibodies specific to core histones, respectively. Special thanks to Drs Gordon Langsley and Robert Walker for critical reading the manuscript and all members of the lab for fruitful discussions., Centre d’Infection et d’Immunité de Lille (CIIL) - U1019 - UMR 8204 (CIIL), Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille)-Centre National de la Recherche Scientifique (CNRS), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Diderot - Paris 7 (UPD7)-Institut de Physique du Globe de Paris (IPG Paris)-Centre National de la Recherche Scientifique (CNRS), and Thomas, Danielle
- Subjects
Proteomics ,MESH: Sequence Analysis, Protein ,Protozoan Proteins ,Antibodies, Protozoan ,MESH: Toxoplasma/pathogenicity ,MESH: DNA-Binding Proteins/chemistry ,Regulatory Sequences, Nucleic Acid ,Mass Spectrometry ,MESH: Protozoan Proteins/chemistry ,Histones ,MESH: Cell Nucleolus/metabolism ,Sequence Analysis, Protein ,MESH: Reverse Transcriptase Polymerase Chain Reaction ,Transcriptional regulation ,MESH: Gene Silencing ,Nuclear protein ,MESH: Ribosomes/metabolism ,Promoter Regions, Genetic ,MESH: High-Throughput Nucleotide Sequencing ,lcsh:QH301-705.5 ,MESH: Promoter Regions, Genetic ,Regulation of gene expression ,MESH: Chromatin Immunoprecipitation ,MESH: Nuclear Proteins/metabolism ,0303 health sciences ,MESH: Histones/metabolism ,Reverse Transcriptase Polymerase Chain Reaction ,MESH: Proteomics ,030302 biochemistry & molecular biology ,High-Throughput Nucleotide Sequencing ,Nuclear Proteins ,Chromatin ,3. Good health ,DNA-Binding Proteins ,MESH: Staining and Labeling ,Histone ,Toxoplasma ,MESH: Nuclear Proteins/genetics ,Cell Nucleolus ,Research Article ,lcsh:Immunologic diseases. Allergy ,Chromatin Immunoprecipitation ,Immunology ,MESH: Protozoan Proteins/genetics ,MESH: Microscopy, Electron ,Biology ,MESH: DNA-Binding Proteins/metabolism ,Microbiology ,MESH: Toxoplasma/metabolism ,Tacrolimus Binding Proteins ,03 medical and health sciences ,Virology ,Genetics ,[SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Nucleosome ,MESH: Antibodies, Protozoan ,MESH: Regulatory Sequences, Nucleic Acid ,[SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology ,Gene Silencing ,Molecular Biology/Chromatin Structure ,Molecular Biology ,MESH: Protozoan Proteins/metabolism ,030304 developmental biology ,MESH: Protozoan Proteins/biosynthesis ,MESH: Mass Spectrometry ,MESH: DNA-Binding Proteins/genetics ,Staining and Labeling ,MESH: Nuclear Proteins/biosynthesis ,Infectious Diseases/Protozoal Infections ,Promoter ,Molecular biology ,MESH: Tacrolimus Binding Proteins/chemistry ,MESH: Cell Nucleolus/genetics ,MESH: Nuclear Proteins/chemistry ,Microscopy, Electron ,lcsh:Biology (General) ,Molecular Biology/Nucleolus and Nuclear Bodies ,MESH: Chromatin/metabolism ,biology.protein ,Parasitology ,MESH: Toxoplasma/genetics ,lcsh:RC581-607 ,Chromatin immunoprecipitation ,Ribosomes - Abstract
In Toxoplasma gondii, cis-acting elements present in promoter sequences of genes that are stage-specifically regulated have been described. However, the nuclear factors that bind to these cis-acting elements and regulate promoter activities have not been identified. In the present study, we performed affinity purification, followed by proteomic analysis, to identify nuclear factors that bind to a stage-specific promoter in T. gondii. This led to the identification of several nuclear factors in T. gondii including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. Using anti-TgNF3 antibodies, HA-FLAG and YFP-tagged TgNF3, we show that TgNF3 is predominantly a parasite nucleolar, chromatin-associated protein that binds specifically to T. gondii gene promoters in vivo. Genome-wide analysis using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) identified promoter occupancies by TgNF3. In addition, TgNF3 has a direct role in transcriptional control of genes involved in parasite metabolism, transcription and translation. The ectopic expression of TgNF3 in the tachyzoites revealed dynamic changes in the size of the nucleolus, leading to a severe attenuation of virulence in vivo. We demonstrate that TgNF3 physically interacts with H3, H4 and H2A/H2B assembled into bona fide core and nucleosome-associated histones. Furthermore, TgNF3 interacts specifically to histones in the context of stage-specific gene silencing of a promoter that lacks active epigenetic acetylated histone marks. In contrast to virulent tachyzoites, which express the majority of TgNF3 in the nucleolus, the protein is exclusively located in the cytoplasm of the avirulent bradyzoites. We propose a model where TgNF3 acts essentially to coordinate nucleolus and nuclear functions by modulating nucleosome activities during the intracellular proliferation of the virulent tachyzoites of T. gondii., Author Summary Apicomplexa including Toxoplasma gondii are responsible for a variety of deadly infections. These intracellular parasites have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression in response to different environments. However, to date, little is known about nuclear factors that regulate their gene expression. Here, we have characterized parasite nuclear factors that bind to a stage-specific promoter. We identified several nuclear factors including a novel factor, designated herein as TgNF3. The N-terminal domain of TgNF3 shares similarities with the N-terminus of yeast nuclear FK506-binding protein (FKBP), known as a histone chaperone regulating gene silencing. We show that TgNF3 is predominantly a nucleolar, chromatin-associated protein that specifically binds to T. gondii nucleosome-associated histones and promoters. Genome-wide analysis identified promoter occupancies by TgNF3 and we demonstrated a direct role for this factor in transcriptional control of genes involved in parasite metabolism, transcription and translation. Ectopic expression of TgNF3 induces dynamic changes in the size of the nucleolus, and a severe attenuation of parasite virulence in vivo. In avirulent bradyzoites, TgNF3 is found exclusively in the cytoplasm, suggesting a potential role in regulating nucleolar and nuclear functions in the virulent tachyzoites of T. gondii.
- Published
- 2011
- Full Text
- View/download PDF