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1.
Curr Biol ; 28(3): 383-391.e3, 2018 02 05.
Article in English | MEDLINE | ID: mdl-29395921

ABSTRACT

The cell fate decision leading to gametogenesis requires the convergence of multiple signals on the promoter of a master regulator. In fission yeast, starvation-induced signaling leads to the transcriptional induction of the ste11 gene, which encodes the central inducer of mating and gametogenesis, known as sporulation. We find that the long intergenic non-coding (linc) RNA rse1 is transcribed divergently upstream of the ste11 gene. During vegetative growth, rse1 directly recruits a Mug187-Lid2-Set1 complex that mediates cis repression at the ste11 promoter through SET3C-dependent histone deacetylation. The absence of rse1 bypasses the starvation-induced signaling and induces gametogenesis in the presence of nutrients. Our data reveal that the remodeling of chromatin through ncRNA scaffolding of repressive complexes that is observed in higher eukaryotes is a conserved, likely very ancient mechanism for tight control of cell differentiation.


Subject(s)
RNA, Fungal/metabolism , RNA, Long Noncoding/metabolism , Schizosaccharomyces/physiology , Ribonucleoprotein, U2 Small Nuclear/genetics , Ribonucleoprotein, U2 Small Nuclear/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
2.
Sci Rep ; 7: 43221, 2017 03 03.
Article in English | MEDLINE | ID: mdl-28256531

ABSTRACT

Human T-lymphotropic Virus type 1 (HTLV-1) infection is characterized by viral latency in the majority of infected cells and by the absence of viremia. These features are thought to be due to the repression of viral sense transcription in vivo. Here, our in silico analysis of the HTLV-1 Long Terminal Repeat (LTR) promoter nucleotide sequence revealed, in addition to the four Sp1 binding sites previously identified, the presence of two additional potential Sp1 sites within the R region. We demonstrated that the Sp1 and Sp3 transcription factors bound in vitro to these two sites and compared the binding affinity for Sp1 of all six different HTLV-1 Sp1 sites. By chromatin immunoprecipitation experiments, we showed Sp1 recruitment in vivo to the newly identified Sp1 sites. We demonstrated in the nucleosomal context of an episomal reporter vector that the Sp1 sites interfered with both the sense and antisense LTR promoter activities. Interestingly, the Sp1 sites exhibited together a repressor effect on the LTR sense transcriptional activity but had no effect on the LTR antisense activity. Thus, our results demonstrate the presence of two new functional Sp1 binding sites in the HTLV-1 LTR, which act as negative cis-regulatory elements of sense viral transcription.


Subject(s)
Epigenetic Repression , Host-Pathogen Interactions , Human T-lymphotropic virus 1/growth & development , Human T-lymphotropic virus 1/genetics , Sp1 Transcription Factor/metabolism , Terminal Repeat Sequences , Transcription, Genetic , Binding Sites , Chromatin Immunoprecipitation , HEK293 Cells , Humans , Jurkat Cells , Protein Binding , Sp3 Transcription Factor/metabolism
3.
Sci Rep ; 6: 31125, 2016 08 22.
Article in English | MEDLINE | ID: mdl-27545598

ABSTRACT

Bovine leukemia virus latency is a viral strategy used to escape from the host immune system and contribute to tumor development. However, a highly expressed BLV micro-RNA cluster has been reported, suggesting that the BLV silencing is not complete. Here, we demonstrate the in vivo recruitment of RNA polymerase III to the BLV miRNA cluster both in BLV-latently infected cell lines and in ovine BLV-infected primary cells, through a canonical type 2 RNAPIII promoter. Moreover, by RPC6-knockdown, we showed a direct functional link between RNAPIII transcription and BLV miRNAs expression. Furthermore, both the tumor- and the quiescent-related isoforms of RPC7 subunits were recruited to the miRNA cluster. We showed that the BLV miRNA cluster was enriched in positive epigenetic marks. Interestingly, we demonstrated the in vivo recruitment of RNAPII at the 3'LTR/host genomic junction, associated with positive epigenetic marks. Functionally, we showed that the BLV LTR exhibited a strong antisense promoter activity and identified cis-acting elements of an RNAPII-dependent promoter. Finally, we provided evidence for an in vivo collision between RNAPIII and RNAPII convergent transcriptions. Our results provide new insights into alternative ways used by BLV to counteract silencing of the viral 5'LTR promoter.


Subject(s)
Genome, Viral , Leukemia Virus, Bovine/enzymology , Leukemia Virus, Bovine/genetics , RNA Polymerase III/genetics , RNA Polymerase II/genetics , 3' Untranslated Regions , Animals , Binding Sites/genetics , Cattle , Cell Line , Epigenesis, Genetic , Gene Knockdown Techniques , HEK293 Cells , Humans , MicroRNAs/genetics , MicroRNAs/metabolism , Promoter Regions, Genetic , Protein Subunits , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , RNA Polymerase III/chemistry , RNA Polymerase III/metabolism , RNA, Small Interfering/genetics , Sheep , Transcription, Genetic
4.
Biochem Soc Trans ; 41(6): 1673-8, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24256273

ABSTRACT

Sexual reproduction is a fundamental aspect of eukaryotic cells, and a conserved feature of gametogenesis is its dependency on a master regulator. The ste11 gene was isolated more than 20 years ago by the Yamamoto laboratory as a suppressor of the uncontrolled meiosis driven by a pat1 mutant. Numerous studies from this laboratory and others have established the role of the Ste11 transcription factor as the master regulator of the switch between proliferation and differentiation in fission yeast. The transcriptional and post-transcriptional controls of ste11 expression are intricate, but most are not redundant. Whereas the transcriptional controls ensure that the gene is transcribed at a high level only when nutrients are rare, the post-transcriptional controls restrict the ability of Ste11 to function as a transcription factor to the G1-phase of the cell cycle from where the differentiation programme is initiated. Several feedback loops ensure that the cell fate decision is irreversible. The complete panel of molecular mechanisms operating to warrant the timely expression of the ste11 gene and its encoded protein basically mirrors the advances in the understanding of the numerous ways by which gene expression can be modulated.


Subject(s)
Gametogenesis , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Transcription Factors/metabolism , Cell Proliferation , Schizosaccharomyces/cytology , Schizosaccharomyces/genetics , Schizosaccharomyces pombe Proteins/genetics , Transcription Factors/genetics , Transcription, Genetic/genetics
5.
Mol Cell Proteomics ; 10(9): M111.008953, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21610105

ABSTRACT

Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.


Subject(s)
Cell Movement , Endoplasmic Reticulum/metabolism , Glycoproteins/chemistry , Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Molecular Motor Proteins/metabolism , Protozoan Proteins/metabolism , Toxoplasma/metabolism , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Movement/physiology , Fibroblasts/cytology , Fibroblasts/parasitology , Glycomics , Glycoproteins/genetics , Glycoproteins/metabolism , Glycosylation , Host-Parasite Interactions/genetics , Humans , Mass Spectrometry , Membrane Proteins/genetics , Molecular Motor Proteins/genetics , Mutagenesis, Site-Directed , Plasmids , Protein Binding , Protein Transport/physiology , Proteomics , Protozoan Proteins/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Toxoplasma/genetics , Transfection
6.
PLoS Pathog ; 7(2): e1001276, 2011 Feb 10.
Article in English | MEDLINE | ID: mdl-21347343

ABSTRACT

Obligate intracellular Apicomplexa parasites share a unique invasion mechanism involving a tight interaction between the host cell and the parasite surfaces called the moving junction (MJ). The MJ, which is the anchoring structure for the invasion process, is formed by secretion of a macromolecular complex (RON2/4/5/8), derived from secretory organelles called rhoptries, into the host cell membrane. AMA1, a protein secreted from micronemes and associated with the parasite surface during invasion, has been shown in vitro to bind the MJ complex through a direct association with RON2. Here we show that RON2 is inserted as an integral membrane protein in the host cell and, using several interaction assays with native or recombinant proteins, we define the region that binds AMA1. Our studies were performed both in Toxoplasma gondii and Plasmodium falciparum and although AMA1 and RON2 proteins have diverged between Apicomplexa species, we show an intra-species conservation of their interaction. More importantly, invasion inhibition assays using recombinant proteins demonstrate that the RON2-AMA1 interaction is crucial for both T. gondii and P. falciparum entry into their host cells. This work provides the first evidence that AMA1 uses the rhoptry neck protein RON2 as a receptor to promote invasion by Apicomplexa parasites.


Subject(s)
Antigens, Protozoan/metabolism , Apicomplexa/physiology , Host-Parasite Interactions/physiology , Protozoan Proteins/metabolism , Virus Internalization , Animals , Antigens, Protozoan/chemistry , Antigens, Protozoan/genetics , Apicomplexa/genetics , Apicomplexa/metabolism , Cells, Cultured , Chlorocebus aethiops , Connexins/metabolism , Conserved Sequence , Host-Parasite Interactions/genetics , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Membrane Proteins/physiology , Models, Biological , Models, Molecular , Parasites/genetics , Parasites/metabolism , Parasites/physiology , Plasmodium falciparum/genetics , Plasmodium falciparum/metabolism , Plasmodium falciparum/physiology , Protein Binding/genetics , Protein Interaction Domains and Motifs/genetics , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Toxoplasma/genetics , Toxoplasma/metabolism , Toxoplasma/physiology , Vero Cells
7.
Mol Cell Proteomics ; 7(5): 891-910, 2008 May.
Article in English | MEDLINE | ID: mdl-18187410

ABSTRACT

The apicomplexan parasite Toxoplasma gondii recognizes, binds, and penetrates virtually any kind of mammalian cell using a repertoire of proteins released from late secretory organelles and a unique form of gliding motility (also named glideosome) that critically depends on actin filaments and myosin. How T. gondii glycosylated proteins mediate host-parasite interactions remains elusive. To date, only limited evidence is available concerning N-glycosylation in apicomplexans. Here we report comprehensive proteomics and glycomics analyses showing that several key components required for host cell-T. gondii interactions are N-glycosylated. Detailed structural characterization confirmed that N-glycans from T. gondii total protein extracts consist of oligomannosidic (Man(5-8)(GlcNAc)2) and paucimannosidic (Man(3-4)(GlcNAc)2) sugars, which are rarely present on mature eukaryotic glycoproteins. In situ fluorescence using concanavalin A and Pisum sativum agglutinin predominantly stained the entire parasite body. Visualization of Toxoplasma glycoproteins purified by affinity chromatography followed by detailed proteomics and glycan analyses identified components involved in gliding motility, moving junction, and other additional functions implicated in intracellular development. Importantly tunicamycin-treated parasites were considerably reduced in motility, host cell invasion, and growth. Collectively these results indicate that N-glycosylation probably participates in modifying key proteins that are essential for host cell invasion by T. gondii.


Subject(s)
Glycomics , Glycoproteins/metabolism , Host-Parasite Interactions , Proteomics , Protozoan Proteins/metabolism , Toxoplasma/physiology , Animals , Carbohydrate Sequence , Cells, Cultured , Glycoproteins/analysis , Glycosylation , Humans , Microscopy, Confocal , Molecular Sequence Data , Oligosaccharides/analysis , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/chemistry , Plant Lectins/chemistry , Polysaccharides/chemistry , Protozoan Proteins/analysis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Toxoplasma/chemistry , Toxoplasma/metabolism
8.
Eukaryot Cell ; 6(9): 1606-17, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17660360

ABSTRACT

We have characterized the Toxoplasma gondii protein phosphatase type 1 (TgPP1) and a potential regulatory binding protein belonging to the leucine-rich repeat protein family, designated TgLRR1. TgLRR1 is capable of binding to TgPP1 to inhibit its activity and to override a G(2)/M cell cycle checkpoint in Xenopus oocytes. In the parasite, TgLRR1 mRNA and protein are both highly expressed in the rapidly replicating and virulent tachyzoites, while only low levels are detected in the slowly dividing and quiescent bradyzoites. TgPP1 mRNA and protein levels are equally abundant in tachyzoites and bradyzoites. Affinity pull down and immunoprecipitation experiments reveal that the TgLRR1-TgPP1 interaction takes place in the nuclear subcompartment of tachyzoites. These results are consistent with those of localization studies using both indirect immunofluorescence with specific polyclonal antibody and transient transfection of T. gondii vector expressing TgLRR1 and TgPP1. The inability to obtain stable transgenic tachyzoites suggested that overexpression of TgLRR1 and TgPP1 may impair the parasite's growth. Together with the activation of Xenopus oocyte meiosis reinitiation, these data indicate that TgLRR1 protein could play a role in the regulation of the T. gondii cell cycle through the modulation of phosphatase activity.


Subject(s)
Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/metabolism , Proteins/metabolism , Protozoan Proteins/metabolism , Toxoplasma/growth & development , Amino Acid Sequence , Animals , Gene Expression , Leucine-Rich Repeat Proteins , Molecular Sequence Data , Oocytes , Phosphoprotein Phosphatases/genetics , Proteins/analysis , Proteins/genetics , Protozoan Proteins/analysis , Protozoan Proteins/genetics , RNA, Messenger/metabolism , Toxoplasma/chemistry , Toxoplasma/metabolism , Xenopus
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