RESUMEN
Histone acetylation, a reversible modification of the core histones, is widely accepted to be involved in remodeling chromatin organization for genetic reprogramming. Histone acetylation is a dynamic process that is regulated by two classes of enzymes, the histone acetyltransferases (HATs) and histone deacetylases (HDACs). Although promoter-specific acetylation and deacetylation has received most of the recent attention, it is superimposed upon a broader acting and dynamic acetylation that profoundly affects many nuclear processes. In this study, we monitored this broader histone acetylation as cells enter and exit mitosis. In contrast to the hypothesis that HATs and HDACs remain bound to mitotic chromosomes to provide an epigenetic imprint for postmitotic reactivation of the genome, we observed that HATs and HDACs are spatially reorganized and displaced from condensing chromosomes as cells progress through mitosis. During mitosis, HATs and HDACs are unable to acetylate or deacetylate chromatin in situ despite remaining fully catalytically active when isolated from mitotic cells and assayed in vitro. Our results demonstrate that HATs and HDACs do not stably bind to the genome to function as an epigenetic mechanism of selective postmitotic gene activation. Our results, however, do support a role for spatial organization of these enzymes within the cell nucleus and their relationship to euchromatin and heterochromatin postmitotically in the reactivation of the genome.
Asunto(s)
Acetiltransferasas/metabolismo , Cromatina/metabolismo , Histona Desacetilasas/metabolismo , Mitosis , Proteínas de Saccharomyces cerevisiae , Acetilación , Animales , Western Blotting , Línea Celular , Histona Acetiltransferasas , Microscopía Fluorescente , FosforilaciónRESUMEN
The cell nucleus is increasingly recognized as a spatially organized structure. In this review, the nature and controversies associated with nuclear compartmentalization are discussed. The relationship between nuclear structure and organization of proteins involved in the regulation of RNA polymerase II-transcribed genes is then discussed. Finally, very recent data on the mobility of these proteins within the cell nucleus is considered and their implications for regulation through compartmentalization of proteins and genomic DNA are discussed.
Asunto(s)
Compartimento Celular , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Acetilación , Cromatina/química , Cromatina/metabolismo , Histonas/metabolismo , Humanos , Interfase , Conformación Proteica , Receptores de Estrógenos/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The transcription coactivator and histone acetyltransferase CAMP response element-binding protein (CBP) has been demonstrated to accumulate in promyelocytic leukemia (PML) bodies. We show that this accumulation is cell type specific. In cells where CBP does not normally accumulate in PML bodies, it can be induced to accumulate in PML bodies through overexpression of either CBP or Pml, but not Sp100. Using fluorescence recovery after photobleaching, we demonstrate that CBP moves rapidly into and out of PML bodies. In contrast, Pml and Sp100 are relatively immobile in the nucleoplasm and within PML nuclear bodies. They possess the characteristics expected of proteins that would play a structural role in the integrity of these subnuclear domains. Our results are consistent with CBP being a dynamic component of PML bodies and that the steady-state level in these structures can be modulated by Pml.
Asunto(s)
Antígenos Nucleares , Estructuras del Núcleo Celular/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Proteínas Nucleares/metabolismo , Transactivadores/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Estructuras del Núcleo Celular/química , Estructuras del Núcleo Celular/efectos de los fármacos , Fluorescencia , Técnica del Anticuerpo Fluorescente , Humanos , Interferones/farmacología , Leucemia Promielocítica Aguda/patología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Matriz Nuclear/química , Matriz Nuclear/efectos de los fármacos , Matriz Nuclear/metabolismo , Proteínas Nucleares/genética , Proteína de la Leucemia Promielocítica , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Transactivadores/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
Histone (de)acetylation is important for the regulation of fundamental biological processes such as gene expression and DNA recombination. Distinct classes of histone deacetylases (HDACs) have been identified, but how they are regulated in vivo remains largely unexplored. Here we describe results demonstrating that HDAC4, a member of class II human HDACs, is localized in the cytoplasm and/or the nucleus. Moreover, we have found that HDAC4 interacts with the 14-3-3 family of proteins that are known to bind specifically to conserved phosphoserine-containing motifs. Deletion analyses suggested that S246, S467, and S632 of HDAC4 mediate this interaction. Consistent with this, alanine substitutions of these serine residues abrogated 14-3-3 binding. Although these substitutions had minimal effects on the deacetylase activity of HDAC4, they stimulated its nuclear localization and thus led to enhanced transcriptional repression. These results indicate that 14-3-3 proteins negatively regulate HDAC4 by preventing its nuclear localization and thereby uncover a novel regulatory mechanism for HDACs.
Asunto(s)
Histona Desacetilasas/metabolismo , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Células 3T3 , Animales , Células COS , Línea Celular , Línea Celular Transformada , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Histona Desacetilasas/genética , Humanos , Factores de Transcripción MEF2 , Ratones , Factores Reguladores Miogénicos , Unión Proteica , Proteínas Represoras/genética , Fracciones Subcelulares , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
All nuclear RNA synthesis is repressed during the mitotic phase of the cell cycle. In addition, RNA polymerase II (RNAP II), nascent RNA and many transcription factors disengage from DNA during mitosis. It has been proposed that mitotic transcription repression and disengagement of factors are due to either mitotic chromatin condensation or biochemical modifications to the transcription machinery. In this study, we investigate the requirement for chromatin condensation in establishing mitotic transcription repression and factor loss, by analyzing transcription and RNAP II localization in mitotic cells infected with herpes simplex virus type 1. We find that virus-infected cells enter mitosis and that mitotic viral DNA is maintained in a nucleosome-free and noncondensed state. Our data show that RNAP II transcription is repressed on cellular genes that are condensed into mitotic chromosomes and on viral genes that remain nucleosome free and noncondensed. Although RNAP II may interact indirectly with viral DNA during mitosis, it remains transcriptionally unengaged. This study demonstrates that mitotic repression of transcription and loss of transcription factors from mitotic DNA can occur independently of nucleosomal chromatin condensation.
Asunto(s)
Cromatina/metabolismo , Silenciador del Gen , Mitosis/genética , Nucleosomas/genética , Transcripción Genética/genética , Ácido Aspártico Endopeptidasas/metabolismo , Bromodesoxiuridina , Proteína Quinasa CDC2/metabolismo , Compartimento Celular , ADN Viral/metabolismo , Colorantes Fluorescentes , Células HeLa , Herpesvirus Humano 1/metabolismo , Humanos , Hibridación in Situ , Interfase/genética , Nucleosomas/metabolismo , Factores de Transcripción/metabolismo , Replicación Viral/genéticaRESUMEN
Compartmentalization of the nucleus is now recognized as an important level of regulation influencing specific nuclear processes. The mechanism of factor organization and the movement of factors in nuclear space have not been fully determined. Splicing factors, for example, have been shown to move in a directed manner as large intact structures from sites of concentration to sites of active transcription, but splicing factors are also thought to exist in a freely diffusible state. In this study, we examined the movement of a splicing factor, ASF, green fluorescent fusion protein (ASF-GFP) using time-lapse microscopy and the technique fluorescence recovery after photobleaching (FRAP). We find that ASF-GFP moves at rates up to 100 times slower than free diffusion when it is associated with speckles and, surprisingly, also when it is dispersed in the nucleoplasm. The mobility of ASF is consistent with frequent but transient interactions with relatively immobile nuclear binding sites. This mobility is slightly increased in the presence of an RNA polymerase II transcription inhibitor and the ASF molecules further enrich in speckles. We propose that the nonrandom organization of splicing factors reflects spatial differences in the concentration of relatively immobile binding sites.
Asunto(s)
Compartimento Celular/fisiología , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Animales , Sitios de Unión , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Línea Celular , Núcleo Celular/ultraestructura , Difusión , Inhibidores Enzimáticos/farmacología , Fluorescencia , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Ratones , Ciervo Muntjac , Fotoquímica , Inhibidores de Proteínas Quinasas , ARN Polimerasa II/antagonistas & inhibidores , Proteínas de Unión al ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Empalme Serina-Arginina , Factores de Transcripción/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacosRESUMEN
We describe a method to image selectively the protein-based architecture in the eukaryotic cell nucleus using nitrogen and phosphorus mapping. In addition, we describe a method to determine total mass as well as stoichiometric relationships between protein and RNA. This method is illustrated using particulate structures in the nucleus called interchromatin granules. In so doing, we demonstrate that these granules contain heterogeneous nuclear RNA, and have an average protein and RNA content of 3.094 and 1.672 MDa, respectively. We also tested the sensitivity of phosphorus detection by exogenously applying purified duplex DNA to the surfaces of thin sections, and have shown that structures as small as single molecules of duplex DNA can be detected in situ using these electron spectroscopic imaging techniques.
Asunto(s)
Núcleo Celular/química , Proteínas Nucleares/análisis , ARN Nuclear/análisis , Animales , Línea Celular , Núcleo Celular/ultraestructura , Cromatina/metabolismo , Cromatina/ultraestructura , Fibroblastos , Microscopía Electrónica/métodos , Ciervo Muntjac , Nitrógeno/metabolismo , Fósforo/metabolismo , Ribosomas/metabolismoRESUMEN
Histones found within transcriptionally competent and active regions of the genome are highly acetylated. Moreover, these highly acetylated histones have very short half-lives. Thus, both histone acetyltransferases and histone deacetylases must enrich within or near these euchromatic regions of the interphase chromatids. Using an antibody specific for highly acetylated histone H3, we have investigated the organization of transcriptionally active and competent chromatin as well as nuclear histone acetyltransferase and deacetylase activities. We observe an exclusion of highly acetylated chromatin around the periphery of the nucleus and an enrichment near interchromatin granule clusters (IGCs). The highly acetylated chromatin is found in foci that may reflect the organization of highly acetylated chromatin into "chromonema" fibers. Transmission electron microscopy of Indian muntjac fibroblast cell nuclei indicates that the chromatin associated with the periphery of IGCs remains relatively condensed, most commonly found in domains containing chromatin folded beyond 30 nm. Using electron spectroscopic imaging, we demonstrate that IGCs are clusters of ribonucleoprotein particles. The individual granules comprise RNA-rich fibrils or globular regions that fold into individual granules. Quantitative analysis of individual granules indicates that they contain variable amounts of RNA estimated between 1.5 and >10 kb. We propose that interchromatin granules are heterogeneous nuclear RNA-containing particles, some of which may be pre-mRNA generated by nearby transcribed chromatin. An intermediary zone between the IGC and surrounding chromatin is described that contains factors with the potential to provide specificity to the localization of sequences near IGCs.