RESUMEN
Chromosomally separated, co-expressed genes can be in spatial proximity, but there is still debate about how this nuclear organization is achieved. Proposed mechanisms include global genome organization, preferential positioning of chromosome territories, or gene-gene sharing of various nuclear bodies. To investigate this question, we selected a set of genes that were co-expressed upon differentiation of human multipotent stem cells. We applied a novel multi-dimensional analysis procedure which revealed that prior to gene expression, the relative position of these genes was conserved in nuclei. Upon stem cell differentiation and concomitant gene expression, we found that co-expressed genes were closer together. In addition, we found that genes in the same 1-µm-diameter neighborhood associated with either the same splicing speckle or to a lesser extent with the same transcription factory. Dispersal of speckles by overexpression of the serine-arginine (SR) protein kinase cdc2-like kinase Clk2 led to a significant drop in the number of genes in shared neighborhoods. We demonstrate quantitatively that the frequencies of speckle and factory sharing can be explained by assuming stochastic selection of a nuclear body within a restricted sub-volume defined by the original global gene positioning present prior to gene expression. We conclude that the spatial organization of these genes is a two-step process in which transcription-induced association with nuclear bodies enhances and refines a pre-existing global organization.
Asunto(s)
Proteínas Nucleares/metabolismo , ARN Polimerasa II/metabolismo , Ribonucleoproteínas/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Cromosomas/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , ARN Polimerasa II/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Ribonucleoproteínas/genética , Factores de Empalme Serina-Arginina , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
We developed an X-ray microscope using partially coherent object illumination instead of previously used quasi-incoherent illumination. The design permitted the incorporation of a cryogenic tilt stage, enabling tomography of frozen-hydrated, intact adherent cells. We obtained three-dimensional reconstructions of mouse adenocarcinoma cells at â¼36-nm (Rayleigh) and â¼70-nm (Fourier ring correlation) resolution, which allowed us to visualize the double nuclear membrane, nuclear pores, nuclear membrane channels, mitochondrial cristae and lysosomal inclusions.
Asunto(s)
Adenocarcinoma/ultraestructura , Microscopía/métodos , Orgánulos/ultraestructura , Animales , Retículo Endoplásmico/ultraestructura , Luz , Lisosomas/ultraestructura , Ratones , Mitocondrias/ultraestructura , Modelos Estructurales , Membrana Nuclear/ultraestructura , Tomografía/métodos , Tomografía de Coherencia Óptica/métodos , Rayos XRESUMEN
We provide a catalog of 3D cryo soft X-ray tomography (cryo-SXT) images obtained from â¼6 to 12µm thick mouse adenocarcinoma cells. Included are multiple representative images of nuclei, nucleoli, nuclear membrane, nuclear membrane channels, mitochondria, lysosomes, endoplasmic reticulum, filaments and plasma membrane, plus three structures not previously described by cryo-SXT, namely Golgi, microvilli and nuclear-membrane blebs. Sections from the 3D cryo-SXT tomograms for all the preceding structures closely resemble those seen by thin-section transmission electron microscopy (TEM). Some structures such as nuclear-membrane channels and nuclear-membrane blebs are more easily detected by cryo-SXT than TEM most likely due to their better contrast and cellular preservation in cryo-SXT combined with the ability to rapidly locate these structures within a full 3D image. We identify and discuss two current limitations in cryo-SXT: variability in image quality and difficulties in detecting weaker contrast structures such as chromatin and various nuclear bodies. Progress on these points is likely to come from the solution of several technical problems in image acquisition, plus the implementation of advanced cryo soft X-ray microscopy approaches such as phase contrast or optical sectioning.
Asunto(s)
Atlas como Asunto , Membrana Celular/ultraestructura , Modelos Biológicos , Orgánulos/ultraestructura , Tomografía por Rayos X/métodos , Animales , Estructuras del Núcleo Celular/ultraestructura , Criopreservación , Imagenología Tridimensional , Ratones , Reproducibilidad de los Resultados , Células Tumorales CultivadasRESUMEN
According to the transcription factory model, localized transcription sites composed of immobilized polymerase molecules transcribe chromatin by reeling it through the transcription site and extruding it to form a surrounding domain of recently transcribed decondensed chromatin. Although transcription sites have been identified in various cells, surrounding domains of recently transcribed decondensed chromatin have not. We report evidence that transcription sites associated with a tandem gene array in mouse cells are indeed surrounded by or adjacent to a domain of decondensed chromatin composed of sequences from the gene array. Formation of this decondensed domain requires transcription and topoisomerase IIalpha activity. The decondensed domain is enriched for the trimethyl H3K36 mark that is associated with recently transcribed chromatin in yeast and several mammalian systems. Consistent with this, chromatin immunoprecipitation demonstrates a comparable enrichment of this mark in transcribed sequences at the tandem gene array. These results provide new support for the pol II factory model, in which an immobilized polymerase molecule extrudes decondensed, transcribed sequences into its surroundings.
Asunto(s)
Cromatina/metabolismo , Histonas/metabolismo , Sitio de Iniciación de la Transcripción , ADN-Topoisomerasas de Tipo II/metabolismo , Modelos Genéticos , Transcripción GenéticaRESUMEN
The repair of DNA double-strand breaks (DSBs) is facilitated by the phosphorylation of H2AX, which organizes DNA damage signaling and chromatin remodeling complexes in the vicinity of the lesion. The disruption of DNA integrity induces an alteration of chromatin architecture that has been proposed to activate the DNA damage transducing kinase ataxia telangiectasia mutated. However, little is known about the physical properties of damaged chromatin. In this study, we use a photoactivatable version of GFP-tagged histone H2B to examine the mobility and structure of chromatin containing DSBs in living cells. We find that chromatin containing DSBs exhibits limited mobility but undergoes an energy-dependent local expansion immediately after DNA damage. The localized expansion observed in real time corresponds to a 30-40% reduction in the density of chromatin fibers in the vicinity of DSBs, as measured by energy-filtering transmission electron microscopy. The observed opening of chromatin occurs independently of H2AX and ATM. We propose that localized adenosine triphosphate-dependent decondensation of chromatin at DSBs establishes an accessible subnuclear environment that facilitates DNA damage signaling and repair.
Asunto(s)
Adenosina Trifosfato/metabolismo , Cromatina/genética , Daño del ADN/genética , Reparación del ADN/genética , ADN/genética , Animales , Células Cultivadas , Cromatina/química , Cromatina/ultraestructura , Posicionamiento de Cromosoma/genética , ADN/ultraestructura , Metabolismo Energético/genética , Femenino , Fibroblastos , Proteínas Fluorescentes Verdes , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Microscopía Electrónica de Transmisión , Transducción de Señal/genéticaRESUMEN
We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.
Asunto(s)
Nucléolo Celular/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Precursores del ARN/metabolismo , Ribosomas/metabolismo , Ubiquitina/metabolismo , Línea Celular , Nucléolo Celular/química , Nucléolo Celular/ultraestructura , Humanos , Proteínas Nucleares/metabolismo , Inhibidores de Proteasoma , Precursores del ARN/análisis , Precursores del ARN/genética , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/metabolismo , ARN Ribosómico 28S/genética , ARN Ribosómico 28S/metabolismo , Ribosomas/genética , Transcripción Genética , Ubiquitina/análisisRESUMEN
Exchange of the glucocorticoid receptor (GR) at promoter target sites provides the only known system in which transcription factor cycling at a promoter is fast, occurring on a time scale of seconds. The mechanism and function of this rapid exchange are unknown. We provide evidence that proteasome activity is required for rapid GR exchange at a promoter. We also show that chaperones, specifically hsp90, stabilize the binding of GR to the promoter, complicating models in which the associated chaperone, p23, has been proposed to induce GR removal. Our results are the first to connect chaperone and proteasome functions in setting the residence time of a transcription factor at a target promoter. Moreover, our results reveal that longer GR residence times are consistently associated with greater transcriptional output, suggesting a new paradigm in which the rate of rapid exchange provides a means to tune transcriptional levels.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Chaperonas Moleculares/metabolismo , Complejos Multienzimáticos/metabolismo , Regiones Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Transcripción Genética , Animales , Benzoquinonas , Línea Celular Tumoral , Corticosterona/metabolismo , Dexametasona/metabolismo , Inhibidores Enzimáticos/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Glucocorticoides/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Lactamas Macrocíclicas , Virus del Tumor Mamario del Ratón/genética , Virus del Tumor Mamario del Ratón/metabolismo , Ratones , Modelos Biológicos , Complejo de la Endopetidasa Proteasomal , Quinonas/metabolismo , Receptores de Glucocorticoides/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de TiempoRESUMEN
Knowledge of tertiary chromatin structure in mammalian interphase chromosomes is largely derived from artificial tandem arrays. In these model systems, light microscope images reveal fibers or beaded fibers after high-density targeting of transactivators to insertional domains spanning several megabases. These images of fibers have lent support to chromonema fiber models of tertiary structure. To assess the relevance of these studies to natural mammalian chromatin, we identified two different approximately 400-kb regions on human chromosomes 6 and 22 and then examined light microscope images of interphase tertiary chromatin structure when the regions were transcriptionally active and inactive. When transcriptionally active, these natural chromosomal regions elongated, yielding images characterized by a series of adjacent puncta or "beads", referred to hereafter as beaded images. These elongated structures required transcription for their maintenance. Thus, despite marked differences in the density and the mode of transactivation, the natural and artificial systems showed similarities, suggesting that beaded images are generic features of transcriptionally active tertiary chromatin. We show here, however, that these images do not necessarily favor chromonema fiber models but can also be explained by a radial-loop model or even a simple nucleosome affinity, random-chain model. Thus, light microscope images of tertiary structure cannot distinguish among competing models, although they do impose key constraints: chromatin must be clustered to yield beaded images and then packaged within each cluster to enable decondensation into adjacent clusters.
Asunto(s)
Cromatina/química , Cromatina/metabolismo , Cromosomas Humanos Par 22/química , Cromosomas Humanos Par 22/metabolismo , Cromosomas Humanos Par 6/química , Cromosomas Humanos Par 6/metabolismo , ADN/química , Secuencia de Bases , Línea Celular , Cromatina/genética , Cromosomas Humanos Par 22/genética , Cromosomas Humanos Par 6/genética , ADN/genética , Humanos , Hibridación Fluorescente in Situ , Interferones/farmacología , Modelos Biológicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Transcripción GenéticaRESUMEN
Although numerous live-cell measurements have shown that transcription factors (TFs) bind chromatin transiently, no measurements of transient binding have been reported at the endogenous response elements (REs) where transcription is normally induced. Here we show that at endogenous REs the transcriptionally productive specific binding of two TFs, p53 and the glucocorticoid receptor (GR), is transient. We also find that the transient residence times of GR at endogenous REs are roughly comparable to those at an artificial, multi-copy array of gene regulatory sites, supporting the use of multi-copy arrays for live-cell analysis of transcription. Finally, we find that at any moment only a small fraction of TF molecules are engaged in transcriptionally productive binding at endogenous REs. The small fraction of bound factors provides one explanation for gene bursting and it also indicates that REs may often be unoccupied, resulting in partial responses to transcriptional signals.