Perichromatin Region Research Articles

Cryoelectron microscopy of vitrified sections: a new challenge for the analysis of functional nuclear architecture

Cryoelectron microscopy of vitrified sections has become a powerful tool for investigating the fine structural features of cellular compartments. In the present study, this approach has been applied in order to explore the ultrastructural morphology of the interphase nucleus in different mammalian cultured cells. Rat hepatoma, Chinese hamster ovary and Potorus kidney cells were cryofixed by high-pressure freezing and the cryosections were examined at low temperature by transmission electron microscopy. Our results show that while the contrast of nuclear structural domains is remarkably homogeneous in hydrated sections, some of them can be recognised due to their characteristic texture. Thus, condensed chromatin appears finely granular and the perichromatin region contains rather abundant fibro-granular elements suggesting the presence of dispersed chromatin fibres and of perichromatin fibrils and granules. The interchromatin space looks homogeneous and interchromatin granules have not been identified under these preparative conditions. In the nucleolus, the most striking feature is the granular component, while the other parts of the nucleolar body, which appear less contrasted, are difficult to resolve. The nuclear envelope is easily recognisable with its regular perinuclear space and nuclear pore complexes. Our observations are discussed in the context of results obtained by other, more conventional electron microscopic methods.

Higher order chromatin architecture in the cell nucleus: on the way from structure to function

The cell nucleus is a highly compartmentalized structure. In this review we describe controversial views on higher order chromatin organization from the level of higher order chromatin domains built up from folded chromatin fibers to the level of chromosome territories and the interchromatin compartment (IC), which harbors non-chromatin nuclear domains, such as interchromatin granule clusters (IGCs visualized in the electron microscope) or splicing factor-containing speckles (visualized by fluorescence microscopy). Emphasis is laid on the definition and functional importance of a nuclear compartment located at the periphery of chromatin domains in direct contact with the IC, termed the perichromatin region (PR). Ongoing experiments to elucidate the topological relationships between PR and IC have provided new insights into the functional interplay between transcription and splicing. As an example, we discuss the structure and nuclear topology of perichromatin fibrils (FPs) contained in the PR and their functional interplay with IGCs/speckles. In addition we discuss the advantages and drawbacks of experimental approaches currently used to study nuclear architecture and function in fixed and living cells.

The functional architecture of the nucleus as analysed by ultrastructural cytochemistry.

Ultrastructural cytochemistry has been, for many years now, a major tool for investigating structure-function relationships in the cell nucleus. It has been essential in approaching the roles which different nuclear structural constituents can play in nuclear functions. This article briefly summarises transmission electron microscopic studies aimed at characterising in situ nuclear architectural domains and their involvement in main nuclear functions, such as DNA replication, hnRNA transcription and pre-mRNA processing. It discusses the importance of ultrastructural cytochemistry in high resolution analyses of intranuclear distribution of chromatin domains and their topological relationships with other structural interphase nuclear constituents. It puts forward the central role of the perichromatin region as a functional nuclear domain. Finally, it attempts to critically evaluate some future applications of ultrastructural investigations of the nucleus and stresses the importance of combining them with light microscopic analyses of living cells.

Condensed chromatin domains in the mammalian nucleus are accessible to large macromolecules.

Most chromatin in interphase nuclei is part of condensed chromatin domains. Previous work has indicated that transcription takes place primarily at the surface of chromatin domains, that is, in the perichromatin region. It is possible that genes inside chromatin domains are silenced due to inaccessibility to macromolecular components of the transcription machinery. We have tested the accessibility of chromatin domains in nuclei of living cells with proteins and dextrans of different molecular sizes. Our results show that chromatin domains are readily accessible to large macromolecules, including proteins with a molecular weight of several hundred kilodaltons. Therefore, the silencing of genes that are incorporated into such domains is not due to the physical inaccessibility of condensed chromatin domains to transcription factors.

Polycomb group gene silencing proteins are concentrated in the perichromatin compartment of the mammalian nucleus.

Human Polycomb group (PcG) proteins are involved in cell-type-dependent epigenetic gene silencing in an evolutionarily conserved manner. We have analysed the subnuclear localisation of these regulatory proteins in two different human cell lines and in rat liver tissue by means of light and electron immunomicroscopy using specific antibodies. We find that the PcG proteins HPC2, HPH1, BMI1 and RING1 are highly concentrated in the perichromatin compartment, situated at the surface of condensed chromatin domains. This compartment was demonstrated earlier to be the nuclear site where most pre-mRNA synthesis takes place. Interestingly, these PcG proteins are virtually absent from the interior of condensed chromatin areas. The present observations therefore show that transcriptionally active and PcG-silenced loci occur within the same spatially limited nuclear domain. Our novel high-resolution data strongly support the idea that epigenetic PcG-mediated gene silencing is a local event, rather than affecting large chromatin domains. In addition to being associated with the perichromatin region, PcG proteins also occur in the interchromatin space. Implications of these observations for higher order chromatin structure and for the mechanisms of PcG-mediated gene silencing are discussed.

Dynamics of nuclear localization sites for COX-2 in vascular endothelial cells.

We investigated the relationships among expression, activity, and spatial organization of cyclooxygenase (COX-1 and COX-2) in endothelial cells from porcine and human cerebral microvessels and from human umbilical vein. In quiescent cells, COX-1 was detected in the perinuclear zone and the cytoplasm, while COX-2 was mainly a nuclear resident possibly connected with the nuclear matrix. COX-2 immunogold labeling was situated in the nuclear envelope, at the nuclear pores, and in connection with the perichromatin regions of the nucleus, considered to be the sites of active transcription. In human endothelial cells transcriptionally activated by interleukin (IL)-1beta, the nucleus remained a major COX-2 localization site during the first 12 h of stimulation, when COX-2 expression was maximally induced. The continuous rise in prostanoid synthesis at 17-23 h of stimulation was associated with COX-2 relocation from the nucleus to the nuclear envelope and the cytoplasm. IL-1beta did not affect COX-1 expression, activity, and localization. COX-2 nuclear localization sites and trafficking between the nucleus and the cytoplasm in endothelial cells may indicate a novel function of COX-2 in regulating gene expression.

Fine Structural in Situ Analysis of Nascent DNA Movement Following DNA Replication

Nascent DNA (newly replicated DNA) was visualized in situ with regard to the position of the previously replicated DNA and to chromatin structure. Localization of nascent DNA at the replication sites can be achieved through pulse labeling of cells with labeled DNA precursors during very short periods of time. We were able to label V79 Chinese Hamster cells for as shortly as 2 min with BrdU; Br-DNA, detected by immunoelectron microscopy, occurs at the periphery of dense chromatin, at individual dispersed chromatin fibers, and within dispersed chromatin areas. In these regions DNA polymerase α was also visualized. After a 5-min BrdU pulse, condensed chromatin also became labeled. When the pulse was followed by a chase, a larger number of gold particles occurred on condensed chromatin. Double-labeling experiments, consisting in first incubating cells with IdU for 20 min, chased for 10 min and then labeled for 5 min with CldU, reveal CldU-labeled nascent DNA on the periphery of condensed chromatin, while previously replicated IdU-labeled DNA has been internalized into condensed chromatin. Altogether, these results show that the sites of DNA replication correspond essentially to perichromatin regions and that the newly replicated DNA moves rapidly from replication sites toward the interior of condensed chromatin areas.

Supramolecular organization of a chromocentric plant nucleus

Cytochemical staining (EDTA procedure for ribonucleoprotein (RNP) and osmium ammine staining), immunocytochemistry (mAbs anti DNA and trimethyl guanosine cap), hypotonic-formaldehyde fixation and in situ hybridization have been used to study the macromolecular architecture of the interphase nucleus of meristematic cells of a dicotyledon plant with chromocentric nuclei, Chirantodendron pentadactylon. The perichromatin region contains extended chromatin loops, perichromatin fibrils as well as typical and immature perichromatin granules. Hypotonic fixation combined with immunolocalization of trimethyl guanosine cap showed that perichromatin fibrils are associated with snURNPs. These results indicate that in the perichromatin region the splicing of newly synthesized pre-mRNA is taking place. Thus, the same functions are localized in the perichromatin region of plant and animal cell nuclei. RNA-containing granules morphologically similar to perichromatin granules, but significantly smaller in size, were found in the interchromatin region. These granules are frequently grouped in clusters. Filaments of extended chromatin pervade the interchromatin region. These observations show that these structures correspond to Lacandonia granules, previously found in some monocotyledons with reticulate nucleus, but not observed in dicotyledons with a chromocentric nucleus. The presence of these granules in both monocotyledons and dicotyledons suggests that they probably constitute an ancestral character of angiosperms. Perichromatin and Lacandonia granules pediculate and irregular in shape corresponding to immature forms are described for the first time in the nucleus of plants. Immunolocalization indicates that the snURNPs are associated with fibrils, interchromatin granules, coiled bodies and nucleolus associated bodies (NABs). Taken as whole these features demonstrate that intense transcription and splicing occur in the ample interchromatin region of meristematic nuclei of Chirantodendron pentadactylon.

Correlation of the changes of the frequency of perichromatin granules with the RNA content of the interchromatin region of uterine cells in normal and ovariectomized rats. A high resolution in situ hybridization and stereological study

The changes in the number of perichromatin granules (PCG) and the alterations in the RNA content of the interchromatin and perichromatin regions caused by ovariectomy and estradiol injection were studied in rat endometrial fibroblast and myometrial muscle cells. Twelve rats were divided in four groups. A group of rats was fixed without any treatment, the other three groups were ovariectomized and processed 21 days after the operation. One of them was studied without further treatment, and two groups were injected intraperitoneally with 20 μg of 17 β-estradiol hemisuccinate and fixed 0.5 and 2 h after the injection. The frequency of PCG was evaluated in preparations stained with EDTA procedure preferential for RNP. The alterations of RNA content were estimated by post-embedding high resolution in situ hybridization using a total DNA probe labeled with biotinilated nucleotides revealed by streptavidin coupled with 10 nm gold grains. Most of the non-nucleolar labeling is associated to RNP containing fibrils. Perichromatin and interchromatin granules are labeled to a lesser extent. Castration brings about a reduction of the number of PCG and of the numerical density of labeling in endometrial fibroblasts. The injection of estradiol causes a rapid increase in both parameters. On the contrary, the frequency of PCG and intensity of labeling of epithelial endometrial cells and in muscle cells increase after ovariectomy and are reduced by estradiol administration. These results suggest that estradiol may affect differentially various types of target cells in the same organ, and also that PCG are not the only nuclear compartment of pre-mRNA or mRNA altered by the changes in estradiol, the RNP containing fibrils located in the perichromatin and in the interchromatin regions are also involved.

Ultrastructural analysis of transcription and splicing in the cell nucleus after bromo-UTP microinjection.

In this study we demonstrate, at an ultrastructural level, the in situ distribution of heterogeneous nuclear RNA transcription sites after microinjection of 5-bromo-UTP (BrUTP) into the cytoplasm of living cells and subsequent postembedding immunoelectron microscopic visualization after different labeling periods. Moreover, immunocytochemical localization of several pre-mRNA transcription and processing factors has been carried out in the same cells. This high-resolution approach allowed us to reveal perichromatin regions as the most important sites of nucleoplasmic RNA transcription and the perichromatin fibrils (PFs) as in situ forms of nascent transcripts. Furthermore, we show that transcription takes place in a rather diffuse pattern, without notable local accumulation of transcription sites. RNA polymerase II, heterogeneous nuclear ribonucleoprotein (hnRNP) core proteins, general transcription factor TFIIH, poly(A) polymerase, splicing factor SC-35, and Sm complex of small nuclear ribonucleoproteins (snRNPs) are associated with PFs. This strongly supports the idea that PFs are also sites of major pre-mRNA processing events. The absence of nascent transcripts, RNA polymerase II, poly(A) polymerase, and hnRNPs within the clusters of interchromatin granules rules out the possibility that this domain plays a role in pre-mRNA transcription and polyadenylation; however, interchromatin granule-associated zones contain RNA polymerase II, TFIIH, and Sm complex of snRNPs and, after longer periods of BrUTP incubation, also Br-labeled RNA. Their role in nuclear functions still remains enigmatic. In the nucleolus, transcription sites occur in the dense fibrillar component. Our fine structural results show that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcriptional and processing events.

Light and Electron Microscope Immunocytochemical Analyses of Histone H1° Distribution in the Nucleus of Friend Erythroleukemia Cells

The localization of histone H1° in murine erythroleukemia cells which were induced to resume a differentiation program was studied in cells which have recovered their proliferative capacity after transient blockage in the G1 phase of the cell cycle. Previous studies have shown that histone H1° accumulation occurs at early times of induction and is probably related to the commitment itself. The distribution of the protein was determined by immunomicroscopy with monoclonal anti-bodies specific for histone H1°. Our observations showed that the histone accumulates in nuclei. Immunoelectron microscopy further demonstrated the presence of histone H1° in condensed chromatin areas, including perinucleolar chromatin. Moreover, histone H1° also occurred in the perichromatin regions, previously described as preferential sites of pre-mRNA synthesis, suggesting that histone H1° is not fully excluded from active chromatin.

Cytochemical and autoradiographic study of the nucleus of a symbiotic dinoflagellate

SUMMARYNon-nucleolar ribonucleoprotein (RNP) particles, extended chromatin, and the distribution of very active sites of transcription are studied in a dinoflagellate symbiont of sea anemones. A preferential staining method for RNP demonstrates granules 47 ± l0nm (standard deviation) in diameter, partially surrounded by a clear halo, and fibres of different thickness. All these particles are sensitive to RNase. Phosphotungstic acid staining for chromatin shows loops of extended chromatin formed by filaments about 7.5 ±3 nm thick protruding from chromosomes. These filaments are sensitive to DNase. Short labelings with tritiated uridine demonstrate a thin perichromosomal region of high transcriptional activity, which may be the functional equivalent to the perichromatin region of higher eukaryotes. In contrast to the eukaryotic perichromatin region, the perichromosomal zone of dinoflagellates has no feature allowing its morphological characterization. A network of filaments 4 ±2nm thick, resistant to RNase ...

Ribonucleoprotein components of root meristematic cell nuclei of the tomato characterized by application of mild loosening and immunocytochemistry

Immunocytochemistry and hypotonic-formaldehyde fixation have been used to study the extranucleolar ribonucleoprotein (RNP) constituents of the nucleus of tomato root meristematic cells. The study of the distribution of small nuclear uridine-rich RNPs (snRNP) by means of a monoclonal anti-Sm antibody recognizing a 29-kDa protein in plants, after standard fixation, shows a preferential labeling of the perichromatin region and a lower labeling of the interchromatin space. These results suggest that in the tomato there is a perichromatin region similar to that of animal cells, in which much of the nonnucleolar transcription and splicing takes place. In hypotonic-formaldehyde—detergent-fixed nuclei, fibrogranular polyparticles have been visualized reacting with anti-snRNP antibody. These structures are frequently associated with filaments of extended chromatin characterized by their reaction with an anti-DNA monoclonal antibody.

Fine structural organization of a non-reticulate plant cell nucleus

We studied the fine structural organization of the meristematic nucleus in roots of Lycopesicon esculentum (tomato) using ultracytochemical and immunocytochemical approaches. The nucleus has a non-reticulate (i.e. low DNA content) structure whose supramolecular organization differs in some respects from that in reticulate nuclei, principally in the organization of the chromocentres associated with the nuclear envelope, with which centromeric structures appear to be associated. The main difference at the nucleolar level is found in the fibrillar centres, which have a low amount of DNA labelling and in which inclusions of condensed chromatin are present only very rarely. The distribution of nucleolar DNA amongst the nucleolar compartments is similar to that in reticulate nucleoli as demonstrated using an anti-DNA monoclonal antibody. Tomato nuclei have nucleolus-associated bodies or karyosomes, like other plant species with a low DNA content and non-reticulate nuclear organization. The nuclear ribonucleoprotein structures in the inter- and perichromatin regions, namely inter- and perichromatin fibrils and granules, show similar ultrastructural and cytochemical characteristics in both types of nuclei.

Ultrastructural immunolocalization of cyclin/PCNA in synchronized 3T3 cells

The immunolocalization of cyclin/PCNA in synchronized 3T3 cells was performed with human autoantibodies using an immunogold technique performed on thin cryosections. Previous immunofluorescent studies demonstrated that the DNA replication sites correspond to the localization of bound cyclin. We have found that in the early periods of S phase, the DNA replication sites (or sites potentially ready for the replication during the hydroxyurea DNA synthesis block) are situated in the perichromatin region and correspond to clustered gold particles present frequently over a morphologically distinct small nuclear area. Heavily labeled chromocenters, including perinucleolar condensed chromatin, exhibiting several such distinct areas were found in later periods of S phase.

Immunoelectron microscopical distribution of poly(ADP-ribose)polymerase in the mammalian cell nucleus

The ultrastructural distribution of poly(ADP-ribose) polymerase has been studied using a specific antibody and immunocytochemistry with immunogold markers. In situ localization in synchronized Chinese hamster ovary cells reveals the antibody associated with mitotic chromosomes, and later with condensed chromatin and perichromatin regions in G 1 phase. During S and G 2, the label occurs mostly on perichromatin regions where perichromatin fibrils are also observed. In the nucleolus, the label appears especially on the dense fibrillar component and to a minor extent on the granular component. Immunolabeled spread active chromatin preparations from exponentially growing cultured mouse P815 cells indicate preferential association of the antibody with nascent nonribosomal RNP fibrils compared to inactive chromatin. The results, suggesting a relationship between the poly(ADP-ribose)polymerase occurrence and RNA (or RNP) formation, are discussed in view of the present knowledge about possible relations between poly(ADP-ribosylation) and synthesis of RNA and DNA.

The Ultrastructural Visualization of Nucleolar and Extranucleolar RNA Synthesis and Distribution

This chapter summarizes current data concerning the ultrastructural localization of transcription sites and the subsequent distribution of newly synthesized ribonucleic acid (RNA) within the cell nucleus. It discusses the roles of ribonucleoprotein (RNP)-containing nuclear structures with respect to the synthesis and processing of nucleolar and extranucleolar RNA. RNA transcription in the nucleolar, as well as extranucleolar, areas takes place predominantly within the regions situated on the border of condensed chromatin. Perichromatin fibrils, considered as the first in situ detected structures containing newly transcribed extranucleolar RNA, originates in these perichromatin regions and the nucleolar fibrillar components are in intimate contact with intranucleolar chromatin areas. DNA replication sites also appear predominantly within this nuclear region. This particular nuclear area consequently plays a key role in the expression of chromatin functions and these findings lead to the conclusion that the layer of chromatin decondensing on the periphery of dense chromatin areas represents the most active chromatin fraction in the nucleus.

Migration of cytoplasmic proteins into nuclei of spleen cells from immunized animals

The kinetics of nuclear labelling in cells exposed to 3H-leucine is examined by means of quantitative ultrastructural autoradiography. Comparison of the rate of cytoplasmic and nuclear labelling suggests that most of the nuclear activity results from the migration of proteins previously elaborated in the cytoplasm. Two-thirds of the nuclear activity is found to be associated with the nucleolus and the perichromatin regions. As these zones are the most active in RNA synthesis, it is suggested that the labelled proteins are related to the formation of ribonucleoproteins, both nucleolar and extranucleolar. A striking similarity was observed in this respect in lymphocytes, blast cells and plasma cells, although their ability in the question of cell division and protein secretion is quite different.