RESUMEN
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
To make the appropriate developmental decisions or maintain homeostasis, cells and organisms must coordinate the expression of their genome and metabolic state. However, the molecular mechanisms that relay environmental cues such as nutrient availability to the appropriate gene expression response remain poorly understood. There is a growing awareness that central components of intermediary metabolism are cofactors or cosubstrates of chromatin-modifying enzymes. As such, their concentrations constitute a potential regulatory interface between the metabolic and chromatin states. In addition, there is increasing evidence for a direct involvement of classic metabolic enzymes in gene expression control. These dual-function proteins may provide a direct link between metabolic programing and the control of gene expression. Here, we discuss our current understanding of the molecular mechanisms connecting metabolism to gene expression and their implications for development and disease.
Asunto(s)
Núcleo Celular/enzimología , Regulación de la Expresión Génica/genética , Metabolismo/genética , Acetilcoenzima A/biosíntesis , Acetilcoenzima A/metabolismo , Animales , Núcleo Celular/metabolismo , Cromatina/química , Cromatina/metabolismo , Histonas/metabolismo , Humanos , NAD/metabolismo , Neoplasias/enzimología , Neoplasias/fisiopatología , Células Madre Pluripotentes/metabolismoRESUMEN
Nucleotide biosynthesis is fundamental to normal cell proliferation as well as to oncogenesis. Tumor suppressor p53, which prevents aberrant cell proliferation, is destabilized through ubiquitylation by MDM2. Ubiquitin-specific protease 7 (USP7) plays a dualistic role in p53 regulation and has been proposed to deubiquitylate either p53 or MDM2. Here, we show that guanosine 5'-monophosphate synthase (GMPS) is required for USP7-mediated stabilization of p53. Normally, most GMPS is sequestered in the cytoplasm, separated from nuclear USP7 and p53. In response to genotoxic stress or nucleotide deprivation, GMPS becomes nuclear and facilitates p53 stabilization by promoting its transfer from MDM2 to a GMPS-USP7 deubiquitylation complex. Intriguingly, cytoplasmic sequestration of GMPS requires ubiquitylation by TRIM21, a ubiquitin ligase associated with autoimmune disease. These results implicate a classic nucleotide biosynthetic enzyme and a ubiquitin ligase, better known for its role in autoimmune disease, in p53 control.
Asunto(s)
Ligasas de Carbono-Nitrógeno/fisiología , Nucleótidos/biosíntesis , Ribonucleoproteínas/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/genética , Neoplasias de la Mama/metabolismo , Ligasas de Carbono-Nitrógeno/análisis , Ligasas de Carbono-Nitrógeno/genética , Ligasas de Carbono-Nitrógeno/metabolismo , Línea Celular Tumoral , Células Cultivadas , Daño del ADN , Drosophila/genética , Femenino , Células HEK293 , Humanos , Ribonucleoproteínas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/fisiología , Peptidasa Específica de Ubiquitina 7 , UbiquitinaciónRESUMEN
Cells need to coordinate gene expression and metabolic state. Inosine monophosphate dehydrogenase (IMPDH) controls the guanine nucleotide pool and, thereby, cell proliferation. We found that Drosophila IMPDH is also a DNA-binding transcriptional repressor. IMPDH attenuates expression of histone genes and E2f, a key driver of cell proliferation. Nuclear IMPDH accumulates during the G2 phase of the cell cycle or following replicative or oxidative stress. Thus, IMPDH can couple the expression of histones and E2F to cellular state. Genome-wide profiling and in vitro binding assays established that IMPDH binds sequence specifically to single-stranded, CT-rich DNA elements. Surprisingly, this DNA-binding function is conserved in E. coli IMPDH. The catalytic function of IMPDH is not required for DNA binding. Yet substitutions that correspond to human retinitis pigmentosa mutations disrupt IMPDH binding to CT-rich, single-stranded DNA elements. By doubling as nucleotide biosynthetic enzyme or transcription factor, IMPDH can either enable or restrict cell proliferation.
Asunto(s)
Ciclo Celular/genética , Proteínas de Drosophila/genética , IMP Deshidrogenasa/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Línea Celular , Núcleo Celular/metabolismo , Proliferación Celular , Inmunoprecipitación de Cromatina , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Factores de Transcripción E2F/genética , Factores de Transcripción E2F/metabolismo , Fase G2/genética , Perfilación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Humanos , IMP Deshidrogenasa/metabolismo , Datos de Secuencia Molecular , Mutación , Unión Proteica , Retinitis Pigmentosa/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Puntos de Control del Ciclo Celular , Ciclina B/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Mitosis , Ciclosoma-Complejo Promotor de la Anafase , Animales , Subunidad Apc11 del Ciclosoma-Complejo Promotor de la Anafase , Proteínas Portadoras/metabolismo , Línea Celular , Ojo Compuesto de los Artrópodos/crecimiento & desarrollo , Ojo Compuesto de los Artrópodos/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular , Silenciador del Gen , Discos Imaginales/metabolismo , Fenotipo , Complejo Represivo Polycomb 1 , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Transcripción Genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitinación , Alas de Animales/crecimiento & desarrolloRESUMEN
Drosophila GMP synthetase binds ubiquitin-specific protease 7 (USP7) and is required for its ability to deubiquitylate histone H2B. Previously, we showed that the GMPS/USP7 complex cooperates with the Polycomb silencing system through removal of the active ubiquitin mark from histone H2B (H2Bub). Here, we explored the interplay between GMPS and USP7 further and assessed their role in hormone-regulated gene expression. Genetic analysis established a strong cooperation between GMPS and USP7, which is counteracted by the histone H2B ubiquitin ligase BRE1. Loss of either GMPS or USP7 led to increased levels of histone H2Bub in mutant animals. These in vivo analyses complement our earlier biochemical results, establishing that GMPS/USP7 mediates histone H2B deubiquitylation. We found that GMPS/USP7 binds ecdysone-regulated loci and that mutants display severe misregulation of ecdysone target genes. Ecdysone receptor (EcR) interacts biochemically and genetically with GMPS/USP7. Genetic and gene expression analyses suggested that GMPS/USP7 acts as a transcriptional corepressor. These results revealed the cooperation between a biosynthetic enzyme and a ubiquitin protease in developmental gene control by hormone receptors.
Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Drosophila melanogaster/genética , Endopeptidasas/metabolismo , Regulación de la Expresión Génica , Receptores de Esteroides/genética , Proteínas Represoras/metabolismo , Animales , Ligasas de Carbono-Nitrógeno/genética , Drosophila melanogaster/enzimología , Ecdisona/metabolismo , Endopeptidasas/genética , Histonas/metabolismo , Proteínas Represoras/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteasas Ubiquitina-EspecíficasRESUMEN
Protein ubiquitylation plays a central role in multiple signal transduction pathways. However, the substrate specificity and potential developmental roles of deubiquitylating enzymes remain poorly understood. Here, we show that the Drosophila ubiquitin protease UBP64 controls cell fate in the developing eye. UBP64 represses neuronal cell fate but promotes the formation of nonneuronal cone cells. Using a proteomics approach, we identified the transcriptional repressor Tramtrack (TTK) as a primary UBP64 substrate. In common with TTK, reduced UBP64 levels lead to a loss of cone cells, supernumerary photoreceptors, and mechanosensory bristle cells. Previously, it was demonstrated that the blockade of neuronal cell fate was relieved by SINA-dependent ubiquitylation and degradation of TTK. We found that UBP64 counteracts SINA function by deubiquitylating TTK, leading to its stabilization and thereby promoting a nonneuronal cell fate. Mass spectrometric mapping revealed that SINA ubiquitylates multiple sites dispersed throughout TTK, which are duly deubiquitylated by UBP64. This observation suggests that both E3 SINA and UBP64 use a scanning mechanism to (de)ubiquitylate TTK. We conclude that the balance of TTK ubiquitylation by SINA and deubiquitylation by UBP64 constitutes a specific posttranslational switch controlling cell fate.
Asunto(s)
Proteínas de Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica , Genes de Insecto , Proteínas Represoras/fisiología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Diferenciación Celular , Núcleo Celular/metabolismo , ADN Complementario , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero , Ojo/embriología , Neuronas/citología , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Células Fotorreceptoras de Invertebrados/citología , Células Fotorreceptoras de Invertebrados/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Represoras/genética , Células Fotorreceptoras Retinianas Conos/citología , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Polycomb group (PcG) epigenetic silencing proteins act through cis-acting DNA sequences, named Polycomb response elements (PREs). Within PREs, Pleiohomeotic (PHO) binding sites and juxtaposed Pc binding elements (PBEs) function as an integrated DNA platform for the synergistic binding of PHO and the multisubunit Polycomb core complex (PCC). Here, we analyzed the architecture of the PHO/PCC/PRE nucleoprotein complex. DNase I footprinting revealed extensive contacts between PHO/PCC and the PRE. Scanning force microscopy (SFM) in combination with DNA topological assays suggested that PHO/PCC wraps the PRE DNA around its surface in a constrained negative supercoil. These features are difficult to reconcile with the simultaneous presence of nucleosomes at the PRE. Indeed, chromatin immunoprecipitations (ChIPs) and nuclease mapping demonstrated that PREs are nucleosome depleted in vivo. We discuss the implications of these findings for models explaining PRE function.
Asunto(s)
Proteínas Represoras/metabolismo , Elementos de Respuesta , Animales , Sitios de Unión/fisiología , Inmunoprecipitación de Cromatina , ADN/química , ADN/metabolismo , Huella de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Epigénesis Genética , Regulación de la Expresión Génica/fisiología , Microscopía de Fuerza Atómica , Modelos Genéticos , Conformación de Ácido Nucleico , Nucleosomas/metabolismo , Proteínas del Grupo Polycomb , Estructura Terciaria de Proteína , Proteínas Represoras/química , Proteínas Represoras/ultraestructura , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
The packaging of eukaryotic genomic DNA into chromatin is modulated through a range of posttranslational histone modifications. Among these, the role of histone ubiquitylation remains poorly understood. Here, we show that the essential Drosophila ubiquitin-specific protease 7 (USP7) contributes to epigenetic silencing of homeotic genes by Polycomb (Pc). We purified USP7 from embryo nuclear extracts as a stable heteromeric complex with guanosine 5'-monophosphate synthetase (GMPS). The USP7-GMPS complex catalyzed the selective deubiquitylation of histone H2B, but not H2A. Biochemical assays confirmed the tight association between USP7 and GMPS in Drosophila embryo extracts. Similar to USP7, mutations in GMPS acted as enhancers of Pc in vivo. USP7 binding to GMPS was required for histone H2B deubiquitylation and strongly augmented deubiquitylation of the human tumor suppressor p53. Thus, GMPS can regulate the activity of a ubiquitin protease. Collectively, these results implicate a biosynthetic enzyme in chromatin control via ubiquitin regulation.