ABSTRACT
Chikungunya virus (CHIKV) is a re-emerging alphavirus that is transmitted to humans by mosquito bites and causes musculoskeletal and joint pain1,2. Despite intensive investigations, the human cellular factors that are critical for CHIKV infection remain unknown, hampering the understanding of viral pathogenesis and the development of anti-CHIKV therapies. Here we identified the four-and-a-half LIM domain protein 1 (FHL1)3 as a host factor that is required for CHIKV permissiveness and pathogenesis in humans and mice. Ablation of FHL1 expression results in the inhibition of infection by several CHIKV strains and o'nyong-nyong virus, but not by other alphaviruses and flaviviruses. Conversely, expression of FHL1 promotes CHIKV infection in cells that do not normally express it. FHL1 interacts directly with the hypervariable domain of the nsP3 protein of CHIKV and is essential for the replication of viral RNA. FHL1 is highly expressed in CHIKV-target cells and is particularly abundant in muscles3,4. Dermal fibroblasts and muscle cells derived from patients with Emery-Dreifuss muscular dystrophy that lack functional FHL15 are resistant to CHIKV infection. Furthermore, CHIKV infection is undetectable in Fhl1-knockout mice. Overall, this study shows that FHL1 is a key factor expressed by the host that enables CHIKV infection and identifies the interaction between nsP3 and FHL1 as a promising target for the development of anti-CHIKV therapies.
Subject(s)
Chikungunya Fever/virology , Chikungunya virus/pathogenicity , Host-Derived Cellular Factors/metabolism , Host-Pathogen Interactions , Intracellular Signaling Peptides and Proteins/metabolism , LIM Domain Proteins/metabolism , Muscle Proteins/metabolism , Animals , Cells, Cultured , Chikungunya Fever/drug therapy , Chikungunya virus/drug effects , Chikungunya virus/genetics , Chikungunya virus/growth & development , Female , Fibroblasts/virology , HEK293 Cells , Host-Derived Cellular Factors/genetics , Humans , Intracellular Signaling Peptides and Proteins/deficiency , Intracellular Signaling Peptides and Proteins/genetics , LIM Domain Proteins/deficiency , LIM Domain Proteins/genetics , Male , Mice , Muscle Proteins/deficiency , Muscle Proteins/genetics , Myoblasts/virology , O'nyong-nyong Virus/growth & development , O'nyong-nyong Virus/pathogenicity , Protein Binding , RNA, Viral/biosynthesis , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Virus ReplicationABSTRACT
The aim of the present study was to understand the biology of unintegrated HIV-1 DNA and reveal the mechanisms involved in its transcriptional silencing. We found that histones are loaded on HIV-1 DNA after its nuclear import and before its integration in the host genome. Nucleosome positioning analysis along the unintegrated and integrated viral genomes revealed major differences in nucleosome density and position. Indeed, in addition to the well-known nucleosomes Nuc0, Nuc1, and Nuc2 loaded on integrated HIV-1 DNA, we also found NucDHS, a nucleosome that covers the DNase hypersensitive site, in unintegrated viral DNA. In addition, unintegrated viral DNA-associated Nuc0 and Nuc2 were positioned slightly more to the 5' end relative to their position in integrated DNA. The presence of NucDHS in the proximal region of the long terminal repeat (LTR) promoter was associated with the absence of RNAPII and of the active histone marks H3K4me3 and H3ac at the LTR. Conversely, analysis of integrated HIV-1 DNA showed a loss of NucDHS, loading of RNAPII, and enrichment in active histone marks within the LTR. We propose that unintegrated HIV-1 DNA adopts a repressive chromatin structure that competes with the transcription machinery, leading to its silencing.
Subject(s)
Chromatin Assembly and Disassembly , DNA, Viral/genetics , HIV Infections/genetics , HIV-1/genetics , Histones/genetics , Nucleosomes/genetics , Virus Integration/genetics , Gene Expression Regulation, Viral , Genome, Viral , HIV Infections/virology , Humans , Terminal Repeat Sequences , Transcription, GeneticABSTRACT
Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. We found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. We conclude that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis.
Subject(s)
Cell Cycle Proteins/metabolism , Chromatids/genetics , Chromosomal Proteins, Non-Histone/metabolism , Drosophila Proteins/metabolism , Nuclear Proteins/metabolism , Nucleosome Assembly Protein 1/metabolism , Protein Phosphatase 2/metabolism , Animals , Cell Cycle Proteins/genetics , Centromere/genetics , Chromatids/ultrastructure , Chromosomal Proteins, Non-Histone/genetics , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Genome, Insect , Mitosis/genetics , Nuclear Proteins/genetics , Nucleosome Assembly Protein 1/genetics , Protein Binding , Protein Phosphatase 2/genetics , CohesinsABSTRACT
Chikungunya is an infectious disease caused by the chikungunya virus (CHIKV), an alphavirus transmitted to humans by Aedes mosquitoes, and for which there is no licensed vaccine nor antiviral treatments. By using a loss-of-function genetic screen, we have recently identified the FHL1 protein as an essential host factor for CHIKV tropism and pathogenesis. FHL1 is highly expressed in muscles cells and fibroblasts, the main CHIKV-target cells. FHL1 interacts with the viral protein nsP3 and plays a critical role in CHIKV genome amplification. Experiments in vivo performed in FHL1-deficient mice have shown that these animals are resistant to infection and do not develop muscular lesions. Altogether these observations, published in the journal Nature [1], show that FHL1 is a key host factor for CHIKV pathogenesis and identify the interaction between FHL1 and nsP3 as a promising target for the development of new antiviral strategies.
Le chikungunya est une maladie infectieuse causée par le virus chikungunya (CHIKV), un alphavirus transmis à l'Homme par les moustiques Aedes et contre lequel il n'existe ni vaccin, ni traitements antiviraux. En utilisant une approche de crible génétique par perte de fonction, nous avons récemment identifié la protéine FHL1 comme un facteur cellulaire essentiel pour le tropisme et la pathogénèse du CHIKV. FHL1 est une molécule présente majoritairement dans les cellules musculaires et les fibroblastes, les cibles privilégiées de CHIKV. FHL1 interagit avec la protéine virale nsP3 et joue un rôle décisif dans le mécanisme d'amplification du génome de CHIKV. Des expériences in vivo chez des souris déficientes pour FHL1 ont montré que ces animaux sont résistants à l'infection et ne développent pas de lésions musculaires. L'ensemble de ces observations publiées dans la revue Nature [1] montrent que FHL1 est un facteur cellulaire clé pour la pathogénèse de CHIKV et identifient l'interaction entre FHL1 et nsp3 comme une cible prometteuse pour le développement de nouvelles stratégies antivirales.
Subject(s)
Chikungunya Fever , Chikungunya virus , Animals , Chikungunya virus/genetics , Intracellular Signaling Peptides and Proteins , LIM Domain Proteins , Mice , Muscle Proteins , Tropism , Viral Nonstructural Proteins , Virus ReplicationABSTRACT
macroH2A (mH2A) is an unusual histone variant consisting of a histone H2A-like domain fused to a large nonhistone region. In this work, we show that histone mH2A represses p300- and Gal4-VP16-dependent polymerase II transcription, and we have dissected the mechanism by which this repression is realized. The repressive effect of mH2A is observed at the level of initiation but not at elongation of transcription, and mH2A interferes with p300-dependent histone acetylation. The nonhistone region of mH2A is responsible for both the repression of initiation of transcription and the inhibition of histone acetylation. In addition, the presence of this domain of mH2A within the nucleosome is able to block nucleosome remodeling and sliding of the histone octamer to neighboring DNA segments by the remodelers SWI/SNF and ACF. These data unambiguously identify mH2A as a strong transcriptional repressor and show that the repressive effect of mH2A is realized on at least two different transcription activation chromatin-dependent pathways: histone acetylation and nucleosome remodeling.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Histone Acetyltransferases/antagonists & inhibitors , Histones/metabolism , Nucleosomes/metabolism , Repressor Proteins/metabolism , Trans-Activators/antagonists & inhibitors , Transcription Factors/antagonists & inhibitors , Transcription, Genetic , Acetylation , Animals , Cell Cycle Proteins/metabolism , DNA Polymerase II/metabolism , Down-Regulation , Histone Acetyltransferases/metabolism , Nuclear Proteins/metabolism , Nucleosomes/chemistry , Protein Structure, Tertiary , Trans-Activators/metabolism , Transcription Factors/metabolism , Xenopus laevis , p300-CBP Transcription FactorsABSTRACT
Adaptation to cold and warm conditions requires dramatic change in gene expression. The acclimatization process of the common carp Cyprinus carpio L. in its natural habitat has been used to study how organisms respond to natural environmental changes. At the cellular level, adaptation to cold condition is accompanied by a dramatic alteration in nucleolar structure and a down regulation of the expression of ribosomal genes. We show that the enrichment of condensed chromatin in winter adapted cells is not correlated with an increase of the heterochromatin marker trimethyl and monomethyl K20H4. However, the expression of the tri methyl K4 H3 and of the variant histone macroH2A is significantly increased during the winter season together with a hypermethylation of CpG residues. Taking into account the properties of macroH2A toward chromatin structure and dynamics and its role in gene repression our data suggest that the increased expression of macroH2A and the hypermethylation of DNA which occurs upon winter-acclimatization plays a major role for the reorganization of chromatin structure and the regulation of gene expression during the physiological adaptation to a colder environment.
Subject(s)
Acclimatization , Carps/physiology , Histones/metabolism , Seasons , Animals , Biomarkers/analysis , Biomarkers/metabolism , Carps/metabolism , Cell Nucleus/chemistry , Cell Nucleus/ultrastructure , DNA Methylation , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Heterochromatin/metabolism , Histones/analysis , Histones/genetics , Liver/cytology , RNA, Messenger/analysis , RNA, Messenger/metabolismABSTRACT
During spermatogenesis, the paternal genome is repackaged into a non-nucleosomal, highly compacted chromatin structure. Bioinformatic analysis revealed that Drosophila sperm chromatin proteins are characterized by a motif related to the high-mobility group (HMG) box, which we termed male-specific transcript (MST)-HMG box. MST77F is a MST-HMG-box protein that forms an essential component of sperm chromatin. The deposition of MST77F onto the paternal genome requires the chaperone function of tNAP, a testis-specific NAP protein. MST77F, in turn, enables the stable incorporation of MST35Ba and MST35Bb into sperm chromatin. Following MST-HMG-box protein deposition, the ATP-dependent chromatin remodeler ISWI mediates the appropriate organization of sperm chromatin. Conversely, at fertilization, maternal ISWI targets the paternal genome and drives its repackaging into de-condensed nucleosomal chromatin. Failure of this transition in ISWI mutant embryos is followed by mitotic defects, aneuploidy, and haploid embryonic divisions. Thus, ISWI enables bi-directional transitions between two fundamentally different forms of chromatin.
Subject(s)
Adenosine Triphosphatases/physiology , Genome, Insect , Testis/ultrastructure , Transcription Factors/physiology , Adenosine Triphosphatases/chemistry , Animals , Chromatin/metabolism , Chromatin Assembly and Disassembly , Chromosomes, Insect/genetics , Chromosomes, Insect/metabolism , Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , Drosophila melanogaster , Histones/chemistry , Histones/metabolism , Male , Mitosis , Protein Binding , Spermatozoa/physiology , Testis/metabolism , Transcription Factors/chemistryABSTRACT
One of the most dramatic forms of chromatin reorganization occurs during spermatogenesis, when the paternal genome is repackaged from a nucleosomal to a protamine-based structure. We assessed the role of the canonical histone chaperone CAF1 in Drosophila spermatogenesis. In this process, CAF1 does not behave as a complex, but its subunits display distinct chromatin dynamics. During histone-to-protamine replacement, CAF1-p180 dissociates from the DNA while CAF1-p75 binds and stays on as a component of sperm chromatin. Association of CAF1-p75 with the paternal genome depends on CAF1-p180 and protamines. Conversely, CAF1-p75 binds protamines and is required for their incorporation into sperm chromatin. Histone removal, however, occurs independently of CAF1 or protamines. Thus, CAF1-p180 and CAF1-p75 function in a temporal hierarchy during sperm chromatin assembly, with CAF1-p75 acting as a protamine-loading factor. These results show that CAF1 subunits mediate the assembly of two fundamentally different forms of chromatin.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin/metabolism , Drosophila Proteins/metabolism , Protamines/metabolism , Retinoblastoma-Binding Protein 4/metabolism , Animals , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Histones/metabolism , Male , Protein Subunits/genetics , Protein Subunits/metabolism , Retinoblastoma-Binding Protein 4/genetics , Spermatozoa/metabolismABSTRACT
Polycomb group (PcG) proteins control development and cell proliferation through chromatin-mediated transcriptional repression. We describe a transcription-independent function for PcG protein Posterior sex combs (PSC) in regulating the destruction of cyclin B (CYC-B). A substantial portion of PSC was found outside canonical PcG complexes, instead associated with CYC-B and the anaphase-promoting complex (APC). Cell-based experiments and reconstituted reactions established that PSC and Lemming (LMG, also called APC11) associate and ubiquitylate CYC-B cooperatively, marking it for proteosomal degradation. Thus, PSC appears to mediate both developmental gene silencing and posttranslational control of mitosis. Direct regulation of cell cycle progression might be a crucial part of the PcG system's function in development and cancer.
Subject(s)
Cell Cycle Checkpoints , Cyclin B/metabolism , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Mitosis , Anaphase-Promoting Complex-Cyclosome , Animals , Apc11 Subunit, Anaphase-Promoting Complex-Cyclosome , Carrier Proteins/metabolism , Cell Line , Compound Eye, Arthropod/growth & development , Compound Eye, Arthropod/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , G2 Phase Cell Cycle Checkpoints , Gene Silencing , Imaginal Discs/metabolism , Phenotype , Polycomb Repressive Complex 1 , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Interaction Domains and Motifs , RNA Interference , Transcription, Genetic , Ubiquitin-Protein Ligase Complexes/metabolism , Ubiquitination , Wings, Animal/growth & developmentABSTRACT
The histone variant H2A.Bbd appeared to be associated with active chromatin, but how it functions is unknown. We have dissected the properties of nucleosome containing H2A.Bbd. Atomic force microscopy (AFM) and electron cryo-microscopy (cryo-EM) showed that the H2A.Bbd histone octamer organizes only approximately 130 bp of DNA, suggesting that 10 bp of each end of nucleosomal DNA are released from the octamer. In agreement with this, the entry/exit angle of the nucleosomal DNA ends formed an angle close to 180 degrees and the physico-chemical analysis pointed to a lower stability of the variant particle. Reconstitution of nucleosomes with swapped-tail mutants demonstrated that the N-terminus of H2A.Bbd has no impact on the nucleosome properties. AFM, cryo-EM and chromatin remodeling experiments showed that the overall structure and stability of the particle, but not its property to interfere with the SWI/SNF induced remodeling, were determined to a considerable extent by the H2A.Bbd docking domain. These data show that the whole H2A.Bbd histone fold domain is responsible for the unusual properties of the H2A.Bbd nucleosome.
Subject(s)
Histones/chemistry , Histones/metabolism , Nucleosomes/metabolism , Amino Acid Sequence , Animals , Binding Sites , Chromatin Assembly and Disassembly , Chromosomal Proteins, Non-Histone/chemistry , Chromosomal Proteins, Non-Histone/metabolism , Cryoelectron Microscopy , DNA/chemistry , DNA/metabolism , Genetic Variation , Histones/genetics , Histones/ultrastructure , In Vitro Techniques , Microscopy, Atomic Force , Molecular Sequence Data , Mutation , Protein Folding , Protein Structure, Quaternary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Xenopus Proteins/chemistry , Xenopus Proteins/genetics , Xenopus Proteins/metabolism , Xenopus Proteins/ultrastructure , Xenopus laevisABSTRACT
A histone variant H2ABbd was recently identified, but its function is totally unknown. Here we have studied the structural and functional properties of nucleosome and nucleosomal arrays reconstituted with this histone variant. We show that H2ABbd can replace the conventional H2A in the nucleosome, but this replacement results in alterations of the nucleosomal structure. The remodeling complexes SWI/SNF and ACF are unable to mobilize the variant H2ABbd nucleosome. However, SWI/SNF was able to increase restriction enzyme access to the variant nucleosome and assist the transfer of variant H2ABbd-H2B dimer to a tetrameric histone H3-H4 particle. In addition, the p300- and Gal4-VP16-activated transcription appeared to be more efficient for H2ABbd nucleosomal arrays than for conventional H2A arrays. The intriguing mechanisms by which H2ABbd affects both nucleosome remodeling and transcription are discussed.