title: A novel Toxoplasma gondii nuclear factor TgNF3 is a dynamic chromatin-associated component, modulator of nucleolar architecture and parasite virulence.
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.
authors.affiliation: Center for Infection and Immunity of Lille, CNRS UMR 8204, INSERM U 1019, Institut Pasteur de Lille, Université Lille Nord de France, Lille, France.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Olguin-Lamas
authors.forename: Edwige
authors.initials: E
authors.lastname: Madec
authors.forename: Agnes
authors.initials: A
authors.lastname: Hovasse
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Werkmeister
authors.forename: Isabelle
authors.initials: I
authors.lastname: Callebaut
authors.forename: Christian
authors.initials: C
authors.lastname: Slomianny
authors.forename: Stephane
authors.initials: S
authors.lastname: Delhaye
authors.forename: Thomas
authors.initials: T
authors.lastname: Mouveaux
authors.forename: Christine
authors.initials: C
authors.lastname: Schaeffer-Reiss
authors.forename: Alain
authors.initials: A
authors.lastname: Van Dorsselaer
authors.forename: Stanislas
authors.initials: S
authors.lastname: Tomavo
mesh_terms: D000913:Antibodies, Protozoan; D002466:Cell Nucleolus / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D059014:High-Throughput Nucleotide Sequencing; D006657:Histones / Q000378:metabolism; D013058:Mass Spectrometry; D008854:Microscopy, Electron; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D040901:Proteomics; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013194:Staining and Labeling; D022021:Tacrolimus Binding Proteins / Q000737:chemistry; D014122:Toxoplasma / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*
chemical_list: D000913:Antibodies, Protozoan; D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D015800:Protozoan Proteins; D022021:Tacrolimus Binding Proteins