Positioning of the NOR-bearing chromosomes in relation to nucleoli in daughter cells after mitosis

It is known that chromosomes occupy non-random positions in the cell nucleus. However, it is not clear to what extent their nuclear positions, together with their neighborhood, are conserved in daughter cells. To address specific aspects of this problem, we used the model of the chromosomes carrying ribosomal genes that are organized in clusters termed Nucleolus Organizer Regions (NORs). We compared the association of chosen NOR-bearing chromosomes (NOR-chromosomes) with nucleoli, as well as the numbers of nucleoli, in the pairs of daughter cells, and established how frequently the daughter cells had equal numbers of the homologs of certain NOR-chromosomes associated with individual nucleoli. The daughter cells typically had different numbers of nucleoli. At the same time, using immuno-FISH with probes for chromosomes 14 and 15 in HeLa cells, we found that the cell pairs with identical combinations appeared significantly more frequently than predicted by the random model. Thus, although the total number of chromosomes associated with nucleoli is variable, our data indicate that the position of the NOR-bearing chromosomes in relation to nucleoli is partly conserved through mitosis.     

Spatial organization of chromosome territories in the interphase nucleus of trisomy 21 cells

In the interphase cell nucleus, chromosomes adopt a conserved and non-random arrangement in subnuclear domains called chromosome territories (CTs). Whereas chromosome translocation can affect CT organization in tumor cell nuclei, little is known about how aneuploidies can impact CT organization. Here, we performed 3D-FISH on control and trisomic 21 nuclei to track the patterning of chromosome territories, focusing on the radial distribution of trisomic HSA21 as well as 11 disomic chromosomes. We have established an experimental design based on cultured chorionic villus cells which keep their original mesenchymal features including a characteristic ellipsoid nuclear morphology and a radial CT distribution that correlates with chromosome size. Our study suggests that in trisomy 21 nuclei, the extra HSA21 induces a shift of HSA1 and HSA3 CTs out toward a more peripheral position in nuclear space and a higher compaction of HSA1 and HSA17 CTs. We posit that the presence of a supernumerary chromosome 21 alters chromosome compaction and results in displacement of other chromosome territories from their usual nuclear position.     

Visualization of chromosome territories in interphase nuclei of ovarian nurse cells in Calliphora erythrocephala Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.     

Chromosome order in HeLa cells changes during mitosis and early G1, but is stably maintained during subsequent interphase stages

Whether chromosomes maintain their nuclear positions during interphase and from one cell cycle to the next has been controversially discussed. To address this question, we performed long-term live-cell studies using a HeLa cell line with GFP-tagged chromatin. Positional changes of the intensity gravity centers of fluorescently labeled chromosome territories (CTs) on the order of several microm were observed in early G1, suggesting a role of CT mobility in establishing interphase nuclear architecture. Thereafter, the positions were highly constrained within a range of approximately 1 microm until the end of G2. To analyze possible changes of chromosome arrangements from one cell cycle to the next, nuclei were photobleached in G2 maintaining a contiguous zone of unbleached chromatin at one nuclear pole. This zone was stably preserved until the onset of prophase, whereas the contiguity of unbleached chromosome segments was lost to a variable extent, when the metaphase plate was formed. Accordingly, chromatin patterns observed in daughter nuclei differed significantly from the mother cell nucleus. We conclude that CT arrangements were stably maintained from mid G1 to late G2/early prophase, whereas major changes of CT neighborhoods occurred from one cell cycle to the next. The variability of CT neighborhoods during clonal growth was further confirmed by chromosome painting experiments.     

Analytical study of repair cells

To establish the differential diagnosis of repair cells from malignant cells, 624 repair cells from 11 cases, 547 cells derived from 8 adenocarcinomas and 593 cells from 8 squamous-cell carcinomas were analyzed, with stress on the morphology of the nucleoli. Such planimetric data as mean cellular areas, mean nuclear areas, mean nucleolar areas, nuclear-cytoplasmic ratios and nucleolar-nuclear ratios were fairly similar among the three cell groups. However, the even distribution of nucleoli in the nuclei, the decrease in C-type nucleoli and nucleoli consistently linked with nucleolus-associated chromatin were noted to be reliable criteria for the diagnosis of repair cells along with such well-known morphologic characteristics as the sheetlike arrangement of repair cells, the lack of isolated forms, the finely granular chromatin pattern and the round, smooth nuclear configuration with a thin nuclear membrane.     

Characterization of nuclear compartments identified by ectopic markers in mammalian cells with distinctly different karyotype

The functional organization of chromatin in cell nuclei is a fundamental question in modern cell biology. Individual chromosomes occupy distinct chromosome territories in interphase nuclei. Nuclear bodies localize outside the territories and colocalize with ectopically expressed proteins in a nuclear subcompartment, the interchromosomal domain compartment. In order to investigate the structure of this compartment in mammalian cells with distinctly different karyotypes, we analyzed human HeLa cells (3n+=71 chromosomes) and cells of two closely related muntjac species, the Chinese muntjac (2n=46 chromosomes) and the Indian muntjac (2n=6/7 chromosomes). The distribution of ectopically expressed intermediate filament proteins (vimentin and cytokeratins) engineered to contain a nuclear localization sequence (NLS) and a nuclear particle forming protein (murine Mx1) fused to a yellow fluorescent protein (YFP) was compared. The proteins were predominantly localized in regions with poor DAPI staining independent of the cells karyotype. In contrast to NLS-vimentin, the NLS-modified cytokeratins were also found close to the nuclear periphery. In Indian muntjac cells, NLS-vimentin colocalized with Mx1-YFP as well as the NLS-cytokeratins. Since the distribution of the ectopically expressed protein markers is similar in cells with distinctly different chromosome numbers, the property of the delineated, limited compartment might indeed depend on chromatin organization.     

Overall DNA methylation and chromatin structure of normal and abnormal X chromosomes

DNA methylation patterns were studied at the chromosome level in normal and abnormal X chromosomes using an anti-5-methylcytosine antibody. In man, except for the late-replicating X of female cells, the labeled chromosome structures correspond to R- and T-bands and heterochromatin. Depending on the cell type, the species, and cell culture conditions, the late-replicating X in female cells appears to be more or less undermethylated. Under normal conditions, the only structures that remain methylated on the X chromosomes correspond to pseudoautosomal regions, which harbor active genes. Thus, active genes are usually hypomethylated but are located in methylated chromatin. Structural rearrangements of the X chromosome, such as t(X;X)(pter;pter), induce a Turner syndrome-like phenotype that is inconsistent with the resulting triple-X constitution. This suggests a position effect controlling gene inactivation. The derivative chromosomes are always late replicating, and their duplicated short arms, which harbor pseudoautosomal regions, replicate later than the normal late-replicating X chromosomes. The compaction or condensation of this segment is unusual, with a halo of chromatin surrounding a hypocondensed chromosome core. The chromosome core is hypomethylated, but the surrounding chromatin is slightly labeled. Thus, unusual DNA methylation and chromatin condensation are associated with the observed position effect. This strengthens the hypothesis that DNA methylation at the chromosome level is associated with both chromatin structure and gene expression.     

Visualization of Chromosome Territories in Interphase Nuclei of Ovarian Nurse Cells in <Emphasis Type="Italic">Calliphora erythrocephala</Emphasis> Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.     

Higher-order genome organization in platypus and chicken sperm and repositioning of sex chromosomes during mammalian evolution

In mammals, chromosomes occupy defined positions in sperm, whereas previous work in chicken showed random chromosome distribution. Monotremes (platypus and echidnas) are the most basal group of living mammals. They have elongated sperm like chicken and a complex sex chromosome system with homology to chicken sex chromosomes. We used platypus and chicken genomic clones to investigate genome organization in sperm. In chicken sperm, about half of the chromosomes investigated are organized non-randomly, whereas in platypus chromosome organization in sperm is almost entirely non-random. The use of genomic clones allowed us to determine chromosome orientation and chromatin compaction in sperm. We found that in both species chromosomes maintain orientation of chromosomes in sperm independent of random or non-random positioning along the sperm nucleus. The distance of loci correlated with the total length of sperm nuclei, suggesting that chromatin extension depends on sperm elongation. In platypus, most sex chromosomes cluster in the posterior region of the sperm nucleus, presumably the result of postmeiotic association of sex chromosomes. Chicken and platypus autosomes sharing homology with the human X chromosome located centrally in both species suggesting that this is the ancestral position. This suggests that in some therian mammals a more anterior position of the X chromosome has evolved independently.     

Three-dimensional analysis of the arrangement of compact chromatin in the nucleus of G0 rat lymphocytes

The arrangement of compact chromatin of G₀ lymphocytes was studied in three-dimensional reconstructions of the ensemble of the chromatin and of individual compact chromatin bodies. Rat spleen was serially cut and sections were contrasted with procedures preferential for DNA. Electron microscopy images were digitized, processed, and displayed using a commercial soft-ware package, complemented by a system for three-dimensional reconstruction and analysis developed by us on an IBM-compatible microcomputer provided with an image acquisition board. The reconstructions showed a continuous layer of compact chromatin in contact with the nuclear envelope that prevents the automatic recognition of individual chromatin clumps. The ensemble of the arrangement of compact chromatin was found to be very similar in different lymphocytes. After morphological filtering procedures, the initial mass was divided into individual bodies of compact chromatin, which were tagged. Most of these bodies contact the nuclear envelope. The number of bodies as well as the number of contacts with the envelope are similar and correspond to a haploid number of chromosomes. The largest body is always the one containing nucleolus-associated chromatin. When the cell has two nucleoli, the nucleolus-associated chromatin bodies contact the envelope in diametrically opposed areas. This feature was also described in rat liver cells. It is concluded that: (a) the individualized compact chromatin bodies do not correspond to an entire chromosome or to a pair of chromosomes; (b) the arrangement of compact chromatin is not identical in each G₀ lymphocyte, but there are patterns that are repeated with limited changes; and (c) there are common features that appear in different cell types of individuals of the same species.     

Dynamic changes of territories 17 and 18 during EBV-infection of human lymphocytes

Interphase chromosomes form distinct spatial domains called chromosome territories (CTs). The arrangement of CTs is non-random and correlated with cellular processes such as differentiation. The purpose of this study is to provide some behavior information of CTs during lymphocyte EBV-infection, which is thought to be a general extra-biological model. Three-dimensional fluorescence in situ hybridization (3D-FISH) was performed on human lymphocytes every 24 h over 96 h periods in EBV-infection. Chromosomes 17 and 18 were selected as target territories for similar size and different gene density. The data indicate that the radial position of territories 17 was altered with time, whereas territories 18 showed relative stable localization. The relative CT volume of CTs 18 to 17 also changed with infection. Our study is the first to examine the timely changes of chromatin positioning and folding in EBV-lymphocyte infection. Dynamic changes in position and folding status of target chromosomes reflected an impact of EBV infection on genome stability.     

Disturbance in function and expression of condensin affects chromosome compaction in HeLa cells

Condensin, a major non-histone protein complex on chromosomes, is responsible for the formation of rod-shaped chromosome in mitosis. A heterodimer composed of SMC2 (structural maintenance of chromosomes) and SMC4 subunits constitutes the core part of condensin. Although extensive studies have been done in yeast, fruit fly and Xenopus to uncover the mechanisms and molecular nature of SMC proteins, little is known about the complex in mammalian cells. We have conducted a series of experiments to unveil the nature of condensin complex in human chromosome formation. The results show that overexpression of the C-terminal domain of SMC subunits disturbs chromosome condensation, leading to formation of swollen chromosomes, while knockdown of SMC subunits severely disturbs mitotic chromosome formation, resulting in chromatin bridges between daughter cells and multiple nuclei in single cells. The salt extraction assay indicates that a fraction of the condensin complex is bound to chromatin in interphase, but most of the condensin bind to chromatin at the onset of mitosis. Thus, disturbance in condensin function or expression affects chromosome condensation and influences mitotic progression.     

Comparison of homologous polytene chromosomes in Phaseolus coccineus embryo suspensor cells: morphological,autoradiographic and cytophotometric analyses

The functional behaviour of unpaired homologous polytene chromosomes (2n=22), was investigated in nuclei of Phaseolus coccineus embryo suspensor cells. Observations were carried out on the morphological level and after ³H-thymidine and ³H-uridine autoradiography. Histone and total protein contents in the chromatin were also investigated. It was shown that corresponding regions of homologous chromosomes may show different functional structures. ³H-thymidine incorporation demonstrated differences between homologues in both DNA synthesis leading to chromosome endoreduplication (polytenization) and DNA amplification (extra DNA synthesis). ³H-uridine autoradiography showed that homologous regions in a given chromosome pair may display three labeling patterns: i) both regions labeled; ii) both regions unlabeled; iii) one region labeled and the other unlabeled. These three states are found to occur in different cells of one and the same embryo suspensor. Differences between homologous chromosome regions were also found in the ratios between DNA and protein contents in their chromatin. These results, which show that the functional activity of homologous chromosomes of the same complement may greatly differ, are discussed in relation to the characteristics of the system investigated.     

Conservation of relative chromosome positioning in normal and cancer cells

Chromosomes exist in the interphase nucleus as individual chromosome territories. It is unclear to what extent chromosome territories occupy particular positions with respect to each other and how structural rearrangements, such as translocations, affect chromosome organization within the cell nucleus. Here we analyze the relative interphase positioning of chromosomes in mouse lymphoma cells compared to normal splenocytes. We show that in a lymphoma cell line derived from an ATM(-/-) mouse, two translocated chromosomes are preferentially positioned in close proximity to each other. The relative position of the chromosomes involved in these translocations is conserved in normal splenocytes. Relative positioning of chromosomes in normal splenocytes is not due to their random distribution in the interphase nucleus and persists during mitosis. These observations demonstrate that the relative arrangement of chromosomes in the interphase nucleus can be conserved between normal and cancer cells and our data support the notion that physical proximity facilitates rearrangements between chromosomes.