RESUMEN
We show that the nuclear architecture of rod photoreceptor cells differs fundamentally in nocturnal and diurnal mammals. The rods of diurnal retinas possess the conventional architecture found in nearly all eukaryotic cells, with most heterochromatin situated at the nuclear periphery and euchromatin residing toward the nuclear interior. The rods of nocturnal retinas have a unique inverted pattern, where heterochromatin localizes in the nuclear center, whereas euchromatin, as well as nascent transcripts and splicing machinery, line the nuclear border. The inverted pattern forms by remodeling of the conventional one during terminal differentiation of rods. The inverted rod nuclei act as collecting lenses, and computer simulations indicate that columns of such nuclei channel light efficiently toward the light-sensing rod outer segments. Comparison of the two patterns suggests that the conventional architecture prevails in eukaryotic nuclei because it results in more flexible chromosome arrangements, facilitating positional regulation of nuclear functions.
Asunto(s)
Núcleo Celular/genética , Retina/citología , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/fisiología , Visión Ocular/fisiología , Animales , Animales no Consanguíneos , Evolución Biológica , Diferenciación Celular , Eucromatina , Heterocromatina , Histonas/metabolismo , Mamíferos , Ratones , Ratones Endogámicos C57BLRESUMEN
Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.
Asunto(s)
Replicación del ADN , Animales , Ratones , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , ADN/genética , Replicación del ADN/fisiología , Epigénesis Genética/fisiología , Genoma/genética , Microscopía/métodosRESUMEN
This article focuses on the role of the interchromatin compartment (IC) in shaping nuclear landscapes. The IC is connected with nuclear pore complexes (NPCs) and harbors splicing speckles and nuclear bodies. It is postulated that the IC provides routes for imported transcription factors to target sites, for export routes of mRNA as ribonucleoproteins toward NPCs, as well as for the intranuclear passage of regulatory RNAs from sites of transcription to remote functional sites (IC hypothesis). IC channels are lined by less-compacted euchromatin, called the perichromatin region (PR). The PR and IC together form the active nuclear compartment (ANC). The ANC is co-aligned with the inactive nuclear compartment (INC), comprising more compacted heterochromatin. It is postulated that the INC is accessible for individual transcription factors, but inaccessible for larger macromolecular aggregates (limited accessibility hypothesis). This functional nuclear organization depends on still unexplored movements of genes and regulatory sequences between the two compartments.
Asunto(s)
Núcleo Celular/metabolismo , Cromatina/metabolismo , Heterocromatina/metabolismo , Humanos , Poro Nuclear/metabolismo , Empalme del ARN/fisiología , ARN Mensajero/metabolismo , Ribonucleoproteínas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genéticaRESUMEN
Transcription regulatory elements (TREs) have been extensively studied on the biochemical level with respect to their interactions with transcription factors (TFs), other DNA segments, and larger protein complexes. In this review, we describe concepts and preliminary experimental evidence for positional changes of TREs within a dynamic, functional nuclear architecture. We suggest a multilayered shell-like chromatin organization of chromatin domain clusters with increasing chromatin compaction levels from the periphery toward the interior with a decondensed transcriptionally active peripheral layer and compact repressed chromatin typically located in the interior. This model organization of nuclear architecture implies a differential accessibility of TFs to targets located in co-aligned active and inactive nuclear compartments (ANC and INC). It is based on evidence that active, easily accessible chromatin (perichromatin region, PR) lines a network of channels (interchromatin compartment, IC) involved in nuclear import-export functions. The IC and PR constitute the ANC, whereas transcriptionally noncompetent chromatin with higher compactness is part of the likely less accessible INC. Preliminary experimental evidence shows an enrichment of active TREs in the ANC and of inactive TREs in the INC suggesting positional changes of TREs between the ANC and INC depending on changes in their functional state.
Asunto(s)
Núcleo Celular , ADN , Nucleosomas , Elementos Reguladores de la Transcripción/genética , Animales , Núcleo Celular/genética , Núcleo Celular/ultraestructura , ADN/genética , ADN/ultraestructura , Humanos , Microscopía Electrónica , Modelos Moleculares , Nucleosomas/genética , Nucleosomas/ultraestructuraRESUMEN
Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data.
Asunto(s)
Núcleo Celular/ultraestructura , Cromatina/ultraestructura , Colorantes Fluorescentes/química , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Animales , Línea Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/ultraestructura , Histonas/genética , Histonas/metabolismo , Humanos , Imagenología Tridimensional/instrumentación , Imagenología Tridimensional/estadística & datos numéricos , Ratones , Microscopía Fluorescente/instrumentaciónRESUMEN
Burkholderia cenocepacia, the causative agent of cepacia syndrome, primarily affects cystic fibrosis patients, often leading to death. In the lung, epithelial cells serve as the initial barrier to airway infections, yet their responses to B. cenocepacia have not been fully investigated. Here, we examined the molecular responses of human airway epithelial cells to B. cenocepacia infection. Infection led to early signaling events such as activation of Erk, Akt, and NF-κB. Further, TNFα, IL-6, IL-8, and IL-1ß were all significantly induced upon infection, but no IL-1ß was detected in the supernatants. Because caspase-1 is required for IL-1ß processing and release, we examined its expression in airway epithelial cells. Interestingly, little to no caspase-1 was detectable in airway epithelial cells. Transfection of caspase-1 into airway epithelial cells restored their ability to secrete IL-1ß following B. cenocepacia infection, suggesting that a deficiency in caspase-1 is responsible, at least in part, for the attenuated IL-1ß secretion.
Asunto(s)
Bronquios/metabolismo , Infecciones por Burkholderia/metabolismo , Burkholderia cenocepacia , Células Epiteliales/metabolismo , Interleucina-1beta/metabolismo , Mucosa Respiratoria/metabolismo , Bronquios/microbiología , Bronquios/patología , Infecciones por Burkholderia/genética , Infecciones por Burkholderia/microbiología , Infecciones por Burkholderia/patología , Caspasa 1/biosíntesis , Caspasa 1/genética , Línea Celular , Citocinas/biosíntesis , Citocinas/genética , Células Epiteliales/microbiología , Células Epiteliales/patología , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Interleucina-1beta/genética , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Mucosa Respiratoria/microbiología , Mucosa Respiratoria/patología , TransfecciónRESUMEN
In line with the intentions of an issue celebrating the 50th anniversary of Human Genetics, we focus on a series of frequently cited studies published in this journal during the 1980s and 1990s. These studies have contributed to the rise of molecular cytogenetics. They yielded evidence that chromosomes occupy distinct territories in the mammalian cell nucleus, first obtained with laser-UV-microbeam experiments and thereafter with chromosome painting, and contributed to the development of interphase cytogenetics and comparative genome hybridization. We provide a personal account of experimental concepts, which were developed by us and others, and describe some of the unforeseeable turns and obstacles, which we had to overcome on the way towards an experimental realization. We conclude with a perspective on current developments and goals of molecular cytogenetics.
Asunto(s)
Cromosomas Humanos , Hibridación Genómica Comparativa , Genética Médica , Interfase/genética , Pintura Cromosómica , Humanos , Rayos UltravioletaRESUMEN
Three-dimensional structured illumination microscopy (3D-SIM) has opened up new possibilities to study nuclear architecture at the ultrastructural level down to the ~100 nm range. We present first results and assess the potential using 3D-SIM in combination with 3D fluorescence in situ hybridization (3D-FISH) for the topographical analysis of defined nuclear targets. Our study also deals with the concern that artifacts produced by FISH may counteract the gain in resolution. We address the topography of DAPI-stained DNA in nuclei before and after 3D-FISH, nuclear pores and the lamina, chromosome territories, chromatin domains, and individual gene loci. We also look at the replication patterns of chromocenters and the topographical relationship of Xist-RNA within the inactive X-territory. These examples demonstrate that an appropriately adapted 3D-FISH/3D-SIM approach preserves key characteristics of the nuclear ultrastructure and that the gain in information obtained by 3D-SIM yields new insights into the functional nuclear organization.
Asunto(s)
Cromatina/ultraestructura , Cromosomas/ultraestructura , Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Animales , Línea Celular Tumoral , Núcleo Celular/ultraestructura , Replicación del ADN/genética , Hibridación Fluorescente in Situ/métodos , Ratones , ARN Largo no Codificante , ARN no Traducido/ultraestructuraRESUMEN
Understanding nuclear architecture is indispensable for understanding the cell-type-dependent orchestration of active and silent genes and other nuclear functions, such as RNA splicing, DNA replication and repair. Yet, while it is now generally agreed that chromosomes in the cell nucleus are organized as chromosome territories, present models of chromosome territory architecture differ widely with respect to the possible functional implications of dynamic changes of this architecture during the cell cycle and terminal cell differentiation.
Asunto(s)
Estructuras Cromosómicas/genética , Modelos Genéticos , Animales , Ensamble y Desensamble de Cromatina , Humanos , Modelos Moleculares , Conformación MolecularRESUMEN
A nonrandom radial nuclear organization of genes has been well documented. This study provides further evidence that radial positioning depends on features of corresponding â¼1 Mbp chromatin domains (CDs), which represent the basic units of higher-order chromatin organization. We performed a quantitative three-dimensional analysis of the radial nuclear organization of three genes located on chromosome 1 in a DG75 Burkitt lymphoma-derived cell line. Quantitative real-time polymerase chain reaction revealed similar transcription levels for the three selected genes, whereas the total expression strength (TES) calculated as the sum of transcription of all genes annotated within a surrounding window of about 1 Mbp DNA differed for each region. Radial nuclear position of the studied CDs correlated with TES, i.e., the domain with the highest TES occupied the most interior position. Positions of CDs with stable TES values were stably maintained even under experimental conditions, resulting in genome-wide changes of the expression levels of many other genes. Our results strongly support the hypothesis that knowledge of the local chromatin environment is essential to predict the radial nuclear position of a gene.
Asunto(s)
Núcleo Celular/ultraestructura , Ensamble y Desensamble de Cromatina/genética , Cromosomas Humanos Par 1/genética , Expresión Génica , Genes/genética , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Imagenología Tridimensional , Hibridación Fluorescente in Situ , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The environmental bacterium Burkholderia cenocepacia causes opportunistic lung infections in immunocompromised individuals, particularly in patients with cystic fibrosis. Infections in these patients are associated with exacerbated inflammation leading to rapid decay of lung function, and in some cases resulting in cepacia syndrome, which is characterized by a fatal acute necrotizing pneumonia and sepsis. B. cenocepacia can survive intracellularly in macrophages by altering the maturation of the phagosome, but very little is known on macrophage responses to the intracellular infection. In this study, we have examined the role of the PI3K/Akt signaling pathway in B. cenocepacia-infected monocytes and macrophages. We show that PI3K/Akt activity was required for NF-κB activity and the secretion of proinflammatory cytokines during infection with B. cenocepacia. In contrast to previous observations in epithelial cells infected with other Gram-negative bacteria, Akt did not enhance IκB kinase or NF-κB p65 phosphorylation, but rather inhibited GSK3ß, a negative regulator of NF-κB transcriptional activity. This novel mechanism of modulation of NF-κB activity may provide a unique therapeutic target for controlling excessive inflammation upon B. cenocepacia infection.
Asunto(s)
Infecciones por Burkholderia/inmunología , Burkholderia cenocepacia/inmunología , Glucógeno Sintasa Quinasa 3/fisiología , Quinasa I-kappa B/fisiología , Mediadores de Inflamación/fisiología , Macrófagos/inmunología , Monocitos/inmunología , Proteínas Proto-Oncogénicas c-akt/fisiología , Animales , Infecciones por Burkholderia/patología , Regulación hacia Abajo/inmunología , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Macrófagos/microbiología , Macrófagos/patología , Ratones , Monocitos/microbiología , Monocitos/patología , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación/inmunología , Transducción de Señal/inmunología , Factor de Transcripción ReIA/antagonistas & inhibidoresRESUMEN
We report for the first time the genomics of a nuclear compartment of the eukaryotic cell. 454 sequencing and microarray analysis revealed the pattern of nucleolus-associated chromatin domains (NADs) in the linear human genome and identified different gene families and certain satellite repeats as the major building blocks of NADs, which constitute about 4% of the genome. Bioinformatic evaluation showed that NAD-localized genes take part in specific biological processes, like the response to other organisms, odor perception, and tissue development. 3D FISH and immunofluorescence experiments illustrated the spatial distribution of NAD-specific chromatin within interphase nuclei and its alteration upon transcriptional changes. Altogether, our findings describe the nature of DNA sequences associated with the human nucleolus and provide insights into the function of the nucleolus in genome organization and establishment of nuclear architecture.
Asunto(s)
Nucléolo Celular/genética , Genoma Humano , Genómica , Cromatina/genética , Células HeLa , HumanosRESUMEN
Chromatin compaction differences may have a strong impact on accessibility of individual macromolecules and macromolecular assemblies to their DNA target sites. Estimates based on fluorescence microscopy with conventional resolution, however, suggest only modest compaction differences (â¼2-10×) between the active nuclear compartment (ANC) and inactive nuclear compartment (INC). Here, we present maps of nuclear landscapes with true-to-scale DNA densities, ranging from <5 to >300 Mbp/µm3. Maps are generated from individual human and mouse cell nuclei with single-molecule localization microscopy at â¼20 nm lateral and â¼100 nm axial optical resolution and are supplemented by electron spectroscopic imaging. Microinjection of fluorescent nanobeads with sizes corresponding to macromolecular assemblies for transcription into nuclei of living cells demonstrates their localization and movements within the ANC and exclusion from the INC.
Asunto(s)
Cromatina , ADN , Humanos , Animales , Ratones , ADN/genética , Núcleo Celular/genética , Cromosomas , Microscopía Fluorescente/métodosRESUMEN
Epigenetic alterations induced by ionizing radiation may contribute to radiation carcinogenesis. To detect relative accumulations or losses of constitutive post-translational histone modifications in chromatin regions surrounding DNA double-strand breaks (DSB), we developed a method based on ion microirradiation and correlation of the signal intensities after immunofluorescence detection of the histone modification in question and the DSB marker γ-H2AX. We observed after ionizing irradiation markers for transcriptional silencing, such as accumulation of H3K27me3 and loss of active RNA polymerase II, at chromatin regions labeled by γ-H2AX. Confocal microscopy of whole nuclei and of ultrathin nuclear sections revealed that the histone modification H3K4me3, which labels transcriptionally active regions, is underrepresented in γ-H2AX foci. While some exclusion of H3K4me3 is already evident at the earliest time amenable to this kind of analysis, the anti-correlation apparently increases with time after irradiation, suggesting an active removal process. Focal accumulation of the H3K4me3 demethylase, JARID1A, was observed at damaged regions inflicted by laser irradiation, suggesting involvement of this enzyme in the DNA damage response. Since no accumulation of the repressive mark H3K9me2 was found at damaged sites, we suggest that DSB-induced transcriptional silencing resembles polycomb-mediated silencing rather than heterochromatic silencing.
Asunto(s)
Cromosomas/efectos de la radiación , Daño del ADN/efectos de la radiación , Silenciador del Gen/efectos de la radiación , Histonas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de la radiación , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/efectos de la radiación , Cromatina/química , Cromatina/genética , Cromosomas/química , Cromosomas/genética , Roturas del ADN de Doble Cadena/efectos de la radiación , Femenino , Técnica del Anticuerpo Fluorescente , Rayos gamma/efectos adversos , Histonas/genética , Humanos , Metilación/efectos de la radiación , Microscopía Confocal , Osteosarcoma/genética , Osteosarcoma/patología , Procesamiento Proteico-Postraduccional/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Proteína 2 de Unión a Retinoblastoma/genética , Proteína 2 de Unión a Retinoblastoma/metabolismo , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
The precise localization of transcribed DNA and resulting RNA is an important aspect of the functional architecture of the nucleus. To this end we have developed a novel in situ hybridization approach in combination with immunoelectron microscopy, using sense and anti-sense RNA probes that are derived from total cellular or cytoplasmic poly(A+) RNA. This new technology is much more gentle than classical in situ hybridization using DNA probes and shows excellent preservation of nuclear structure. Carried out on ultrathin sections of fixed and resin-embedded COS-7 cells, it revealed at high resolution the localization of the genes that code for the cellular mRNAs. Quantitative analysis shows that most transcribed DNA is concentrated in the perichromatin region, i.e. the interface between subchromosomal compact chromatin domains and the interchromatin space essentially devoid of DNA. The RNA that is produced is found mainly in the perichromatin region and the interchromatin space. These results imply that in the mammalian nucleus the chromatin fiber is folded so that active genes are predominantly present in the perichromatin region, which is the most prominent site of transcription.
Asunto(s)
Núcleo Celular/química , Cromatina/química , ADN/análisis , Transcripción Genética , Animales , Células COS , Chlorocebus aethiops , Hibridación in Situ/métodos , ARN/biosíntesis , Sondas ARNRESUMEN
A new experimental approach was designed to test different predictions of current models of the nuclear architecture with respect to the topography of transcription. We constructed a plasmid, termed pIndi, which carries a reporter gene coding for a red cytoplasmic fluorescent reporter protein. Transcription of the reporter gene is regulated by the inducible promoter of the human immunodeficiency virus (HIV) and is strongly dependent on the HIV-1 Tat protein. Expressing the red fluorescent reporter protein allowed us to distinguish between cells with active and silent reporter genes. Importantly, transient transfection resulted in the clustering of plasmids, forming one or several extra-chromosomal pIndi bodies. Repetitive lac operator sequences in pIndi allowed us to visualize these bodies in living cells by the binding of LacI proteins tagged with a fluorescent protein. Using this model, we analyzed the three-dimensional nuclear topography of pIndi bodies with active or silent reporter genes. Our results are compatible with predictions of the chromosome territory-interchromatin compartment (CT-IC) model. We demonstrate that pIndi bodies localize in the IC, both in the silent and active state. Activation of transgene transcription resulted in the recruitment of RNA polymerase II and NFkappaB and a closer positioning to splicing speckles.
Asunto(s)
Núcleo Celular/genética , Genes Reporteros , Imagenología Tridimensional/métodos , Transcripción Genética , Animales , Línea Celular , Cromosomas , Técnica del Anticuerpo Fluorescente , Humanos , Métodos , Ratones , FN-kappa B/metabolismo , Plásmidos , Transporte de Proteínas , ARN Polimerasa II/metabolismo , TransgenesRESUMEN
Extensive changes of higher order chromatin arrangements can be observed during prometaphase, terminal cell differentiation and cellular senescence. Experimental systems where major reorganization of nuclear architecture can be induced under defined conditions, may help to better understand the functional implications of such changes. Here, we report on profound chromatin reorganization in fibroblast nuclei by chaetocin, a thiodioxopiperazine metabolite. Chaetocin induces strong condensation of chromosome territories separated by a wide interchromatin space largely void of DNA. Cell viability is maintained irrespective of this peculiar chromatin phenotype. Cell cycle markers, histone signatures, and tests for cellular senescence and for oxidative stress indicate that chaetocin induced chromatin condensation/clustering (CICC) represents a distinct entity among nuclear phenotypes associated with condensed chromatin. The territorial organization of entire chromosomes is maintained in CICC nuclei; however, the conventional nuclear architecture harboring gene-dense chromatin in the nuclear interior and gene-poor chromatin at the nuclear periphery is lost. Instead gene-dense and transcriptionally active chromatin is shifted to the periphery of individual condensed chromosome territories where nascent RNA becomes highly enriched around their outer surface. This chromatin reorganization makes CICC nuclei an attractive model system to study this border zone as a distinct compartment for transcription. Induction of CICC is fully inhibited by thiol-dependent antioxidants, but is not related to the production of reactive oxygen species. Our results suggest that chaetocin functionally impairs the thioredoxin (Trx) system, which is essential for deoxynucleotide synthesis, but in addition involved in a wide range of cellular functions. The mechanisms involved in CICC formation remain to be fully explored.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/fisiología , Antioxidantes/farmacología , Ciclo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Células Cultivadas , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Histonas/metabolismo , Humanos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Piperazinas/metabolismo , Piperazinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Tiorredoxinas/metabolismo , Transcripción Genética , Inactivación del Cromosoma XRESUMEN
Epigenetic changes, including DNA methylation patterns, histone modifications and histone variants, as well as chromatin remodelling play a fundamental role in the regulation of pre- and postimplantation mammalian development. Recent studies have indicated that nuclear architecture provides an additional level of regulation, which needs to be explored in order to understand how a fertilised egg is able to develop into a full organism. Studies of 3D preserved nuclei of IVF preimplantation embryos from different mammalian species, such as mouse, rabbit and cow, have demonstrated that nuclear architecture undergoes major changes during early development. Both similarities and species-specific differences were observed. Nuclear transfer experiments demonstrated changes of nuclear phenotypes, which to some extent reflect changes seen in IVF preimplantation embryos albeit with a different timing compared with IVF embryos. The dynamics of nuclear architecture is further substantiated by major changes during postmitotic terminal cell differentiation. Recent breakthroughs of 3D fluorescence microscopy with resolution beyond the conventional Abbe limit in combination with 3D electron microscopy provide the potential to explore the topography of nuclear structure with unprecedented resolution and detail.
Asunto(s)
Blastocisto/ultraestructura , Diferenciación Celular , Núcleo Celular/ultraestructura , Animales , Blastocisto/citología , Bovinos , Cromosomas/ultraestructura , Ratones , Microscopía Electrónica/métodos , Modelos Animales , Nanoestructuras/ultraestructura , ConejosRESUMEN
Pericentric heterochromatin plays an important role in epigenetic gene regulation. We show that pericentric heterochromatin aggregates during myogenic differentiation. This clustering leads to the formation of large chromocenters and correlates with increased levels of the methyl CpG-binding protein MeCP2 and pericentric DNA methylation. Ectopic expression of fluorescently tagged MeCP2 mimicked this effect, causing a dose-dependent clustering of chromocenters in the absence of differentiation. MeCP2-induced rearrangement of heterochromatin occurred throughout interphase, did not depend on the H3K9 histone methylation pathway, and required the methyl CpG-binding domain (MBD) only. Similar to MeCP2, another methyl CpG-binding protein, MBD2, also increased during myogenic differentiation and could induce clustering of pericentric regions, arguing for functional redundancy. This MeCP2- and MBD2-mediated chromatin reorganization may thus represent a molecular link between nuclear genome topology and the epigenetic maintenance of cellular differentiation.
Asunto(s)
Diferenciación Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Heterocromatina/metabolismo , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo , Proteínas Represoras/metabolismo , Animales , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Metilación de ADN , Proteínas de Unión al ADN/genética , Epigénesis Genética/genética , Regulación del Desarrollo de la Expresión Génica/genética , Heterocromatina/genética , Heterocromatina/ultraestructura , Histonas/genética , Histonas/metabolismo , Masculino , Proteína 2 de Unión a Metil-CpG , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Esquelético/ultraestructura , Mioblastos Esqueléticos/ultraestructura , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genéticaRESUMEN
The nuclear architecture is considered an important contributor to genome function. Although the fine structural features of the cell nucleus have been investigated extensively by means of ultrastructural cytochemistry, mainly on ultrathin sections in two dimensions (2D), there was a of lack routine methods for a rapid reconstruction of three-dimensional (3D) distribution of different structural constituents throughout the nuclear volume. We have now filled this gap by the application of a novel approach associating a pre-embedding selective visualization of nuclear components with a method making use of ultramicrotomy combined with scanning electron microscopy (microtome serial block face scanning electron microscopy--'3View'). We have been able to apply this method to the study of DNA distribution within the nuclear volume and reconstruction of 3D chromatin arrangement in nuclei of rat hepatocytes and endothelial cells. Our observations demonstrate that while chromatin appears to occupy the interior of nuclei rather sparsely on 2D images, once reconstructed in 3D from a series of sequential 2D images it gives the impression of considerably filling the nuclear volume. However, quantitative evaluation of the nuclear volume occupied by DNA in the above two types of nuclei leaves a significant part to the interchromatin space (66.2% for hepatic cells and 41.7% for endothelial cells, including nuclear space occupied by nucleoli). Detailed analysis of the reconstructed nuclei reveals a high degree of superposition of chromatin domains, giving rise to a false impression that they fill a much larger part of the nuclear volume than they really do. Our results show the importance of the contribution of such reconstruction techniques to our understanding of the nuclear architecture.