RESUMO
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.
Assuntos
Montagem e Desmontagem da Cromatina , DNA Viral/genética , Infecções por HIV/genética , HIV-1/genética , Histonas/genética , Nucleossomos/genética , Integração Viral/genética , Regulação Viral da Expressão Gênica , Genoma Viral , Infecções por HIV/virologia , Humanos , Sequências Repetidas Terminais , Transcrição GênicaRESUMO
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.
Assuntos
Adenosina Trifosfatases/fisiologia , Genoma de Inseto , Testículo/ultraestrutura , Fatores de Transcrição/fisiologia , Adenosina Trifosfatases/química , Animais , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Cromossomos de Insetos/genética , Cromossomos de Insetos/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Histonas/química , Histonas/metabolismo , Masculino , Mitose , Ligação Proteica , Espermatozoides/fisiologia , Testículo/metabolismo , Fatores de Transcrição/químicaRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 de Modelagem do Nucleossomo/metabolismo , Proteína Fosfatase 2/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Centrômero/genética , Cromátides/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Genoma de Inseto , Mitose/genética , Proteínas Nucleares/genética , Proteína 1 de Modelagem do Nucleossomo/genética , Ligação Proteica , Proteína Fosfatase 2/genética , CoesinasRESUMO
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.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Protaminas/metabolismo , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Histonas/metabolismo , Masculino , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteína 4 de Ligação ao Retinoblastoma/genética , Espermatozoides/metabolismoRESUMO
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.
