RESUMEN
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.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 de Ensamblaje de Nucleosomas/metabolismo , Proteína Fosfatasa 2/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Centrómero/genética , Cromátides/ultraestructura , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Genoma de los Insectos , Mitosis/genética , Proteínas Nucleares/genética , Proteína 1 de Ensamblaje de Nucleosomas/genética , Unión Proteica , Proteína Fosfatasa 2/genética , CohesinasRESUMEN
Ubiquitin-specific protease 7 (USP7) has been implicated in cancer progression and neurodevelopment. However, its molecular targets remain poorly characterized. We combined quantitative proteomics, transcriptomics, and epigenomics to define the core USP7 network. Our multi-omics analysis reveals USP7 as a control hub that links genome regulation, tumor suppression, and histone H2A ubiquitylation (H2AK119ub1) by noncanonical Polycomb-repressive complexes (ncPRC1s). USP7 strongly stabilizes ncPRC1.6 and, to a lesser extent, ncPRC1.1. Moreover, USP7 represses expression of AUTS2, which suppresses H2A ubiquitylation by ncPRC1.3/5. Collectively, these USP7 activities promote the genomic deposition of H2AK119ub1 by ncPRC1, especially at transcriptionally repressed loci. Notably, USP7-dependent changes in H2AK119ub1 levels are uncoupled from H3K27me3. Even complete loss of the PRC1 catalytic core and H2AK119ub1 has only a limited effect on H3K27me3. Besides defining the USP7 regulome, our results reveal that H2AK119ub1 dosage is largely disconnected from H3K27me3.
RESUMEN
ATP-dependent chromatin remodelers control the accessibility of genomic DNA through nucleosome mobilization. However, the dynamics of genome exploration by remodelers, and the role of ATP hydrolysis in this process remain unclear. We used live-cell imaging of Drosophila polytene nuclei to monitor Brahma (BRM) remodeler interactions with its chromosomal targets. In parallel, we measured local chromatin condensation and its effect on BRM association. Surprisingly, only a small portion of BRM is bound to chromatin at any given time. BRM binds decondensed chromatin but is excluded from condensed chromatin, limiting its genomic search space. BRM-chromatin interactions are highly dynamic, whereas histone-exchange is limited and much slower. Intriguingly, loss of ATP hydrolysis enhanced chromatin retention and clustering of BRM, which was associated with reduced histone turnover. Thus, ATP hydrolysis couples nucleosome remodeling to remodeler release, driving a continuous transient probing of the genome.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Transactivadores/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Línea Celular , Drosophila melanogaster/genética , Histonas/metabolismo , Hidrólisis , Nucleosomas/metabolismoRESUMEN
Polycomb group (PcG) proteins function through cis-acting DNA elements called PcG response elements (PREs) to stably silence developmental regulators, including the homeotic genes. However, the mechanism by which they are targeted to PREs remains largely unclear. Pleiohomeotic (PHO) is a sequence-specific DNA-binding PcG protein and therefore may function to tether other PcG proteins to the DNA. Here, we show that PHO can directly bind to a Polycomb (PC)-containing complex as well as the Brahma (BRM) chromatin-remodeling complex. PHO contacts the BRM complex through its zinc finger DNA-binding domain and a short N-terminal region. A distinct domain of PHO containing a conserved motif contacts the PcG proteins PC and Polyhomeotic (PH). With mobility shift assays and DNA pulldown experiments, we demonstrated that PHO is able to link PC, which lacks sequence-specific DNA-binding activity, to the DNA. Importantly, we found that the PC-binding domain of PHO can mediate transcriptional repression in transfected Drosophila Schneider cells. Concomitant overexpression of PC resulted in stronger PHO-directed repression that was dependent on its PC-binding domain. Together, these results suggest that PHO can contribute to PRE-mediated silencing by direct recruitment of a PC complex to repress transcription.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/fisiología , ADN/metabolismo , Proteínas de Drosophila/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Animales , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Drosophila , Silenciador del Gen , Glutatión Transferasa/metabolismo , Insectos , Modelos Biológicos , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Factores de Transcripción , TransfecciónRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Genoma de los Insectos , Testículo/ultraestructura , Factores de Transcripción/fisiología , Adenosina Trifosfatasas/química , Animales , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Cromosomas de Insectos/genética , Cromosomas de Insectos/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Histonas/química , Histonas/metabolismo , Masculino , Mitosis , Unión Proteica , Espermatozoides/fisiología , Testículo/metabolismo , Factores de Transcripción/químicaRESUMEN
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.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Protaminas/metabolismo , Proteína 4 de Unión a Retinoblastoma/metabolismo , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Histonas/metabolismo , Masculino , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteína 4 de Unión a Retinoblastoma/genética , Espermatozoides/metabolismoRESUMEN
The nucleosome is the fundamental repeating unit of eukaryotic chromatin. Here, we assessed the interplay between DNA sequence and ATP-dependent chromatin-remodeling factors (remodelers) in the nucleosomal organization of a eukaryotic genome. We compared the genome-wide distribution of Drosophila NURD, (P)BAP, INO80, and ISWI, representing the four major remodeler families. Each remodeler has a unique set of genomic targets and generates distinct chromatin signatures. Remodeler loci have characteristic DNA sequence features, predicted to influence nucleosome formation. Strikingly, remodelers counteract DNA sequence-driven nucleosome distribution in two distinct ways. NURD, (P)BAP, and INO80 increase histone density at their target sequences, which intrinsically disfavor positioned nucleosome formation. In contrast, ISWI promotes open chromatin at sites that are propitious for precise nucleosome placement. Remodelers influence nucleosome organization genome-wide, reflecting their high genomic density and the propagation of nucleosome redistribution beyond remodeler binding sites. In transcriptionally silent early embryos, nucleosome organization correlates with intrinsic histone-DNA sequence preferences. Following differential expression of the genome, however, this relationship diminishes and eventually disappears. We conclude that the cellular nucleosome landscape is the result of the balance between DNA sequence-driven nucleosome placement and active nucleosome repositioning by remodelers and the transcription machinery.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ensamble y Desensamble de Cromatina , ADN/metabolismo , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/genética , Animales , Sitios de Unión/genética , ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Genoma , Histonas/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Nucleosomas/genética , Nucleosomas/metabolismo , Factores de Transcripción/genéticaAsunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Adenosina Trifosfato/metabolismo , Cromatina/fisiología , Cromatina/ultraestructura , Cromatografía/métodos , Cromatografía de Afinidad/métodos , Ditiotreitol/farmacología , Escherichia coli/genética , Escherichia coli/ultraestructura , Indicadores y Reactivos , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
SWI/SNF ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in eukaryotic gene expression control. BAP and PBAP are the fly representatives of the two evolutionarily conserved major subclasses of SWI/SNF remodelers. Both complexes share seven core subunits, including the Brahma ATPase, but differ in a few signature subunits; POLYBROMO and BAP170 specify PBAP, whereas OSA defines BAP. Here, we show that the transcriptional coactivator and PHD finger protein SAYP is a novel PBAP subunit. Biochemical analysis established that SAYP is tightly associated with PBAP but absent from BAP. SAYP, POLYBROMO, and BAP170 display an intimately overlapping distribution on larval salivary gland polytene chromosomes. Genome-wide expression analysis revealed that SAYP is critical for PBAP-dependent transcription. SAYP is required for normal development and interacts genetically with core- and PBAP-selective subunits. Genetic analysis suggested that, like BAP, PBAP also counteracts Polycomb silencing. SAYP appears to be a key architectural component required for the integrity and association of the PBAP-specific module. We conclude that SAYP is a signature subunit that plays a major role in the functional specificity of the PBAP holoenzyme.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Factores de Transcripción/fisiología , Animales , Células Cultivadas , Embrión no Mamífero , Larva , Subunidades de Proteína/fisiología , Glándulas Salivales/metabolismo , Transcripción GenéticaRESUMEN
Transcription regulation involves enzyme-mediated changes in chromatin structure. Here, we describe a novel mode of histone crosstalk during gene silencing, in which histone H2A monoubiquitylation is coupled to the removal of histone H3 Lys 36 dimethylation (H3K36me2). This pathway was uncovered through the identification of dRING-associated factors (dRAF), a novel Polycomb group (PcG) silencing complex harboring the histone H2A ubiquitin ligase dRING, PSC and the F-box protein, and demethylase dKDM2. In vivo, dKDM2 shares many transcriptional targets with Polycomb and counteracts the histone methyltransferases TRX and ASH1. Importantly, cellular depletion and in vitro reconstitution assays revealed that dKDM2 not only mediates H3K36me2 demethylation but is also required for efficient H2A ubiquitylation by dRING/PSC. Thus, dRAF removes an active mark from histone H3 and adds a repressive one to H2A. These findings reveal coordinate trans-histone regulation by a PcG complex to mediate gene repression.