Chromosomes are predominantly located randomly with respect to each other in interphase human cells

To test quantitatively whether there are systematic chromosome-chromosome associations within human interphase nuclei, interchanges between all possible heterologous pairs of chromosomes were measured with 24-color whole-chromosome painting (multiplex FISH), after damage to interphase lymphocytes by sparsely ionizing radiation in vitro. An excess of interchanges for a specific chromosome pair would indicate spatial proximity between the chromosomes comprising that pair. The experimental design was such that quite small deviations from randomness (extra pairwise interchanges within a group of chromosomes) would be detectable. The only statistically significant chromosome cluster was a group of five chromosomes previously observed to be preferentially located near the center of the nucleus. However, quantitatively, the overall deviation from randomness within the whole genome was small. Thus, whereas some chromosome-chromosome associations are clearly present, at the whole-chromosomal level, the predominant overall pattern appears to be spatially random.     

Position of chromosomes in the human interphase nucleus

Summary The problem of localization of chromosomes in relation to each other in the interphase nucleus of human lymphocytes was investigated by analysis of chromatid and chromosome aberrations observed in lymphocyte cultures of three patients with Fanconi's anemia, one patient with Bloom's syndrome, and in Trenimon-treated (Trenimon, Bayer) normal cells. Distribution of open gaps and breaks is highly correlated with chromosome length and distribution of breaks involved in chromatid translocations in Fanconi's anemia and in Trenimontreated cells. Both correlations are much lower in Bloom's syndrome. In Fanconi's anemia and in normal cells after Trenimon-treatment, the majority of chromatid translocations are between nonhomologous chromosomes, whereas in Bloom's syndrome mainly homologous chromosomes are involved. Statistical localization of chromosomes in relation to each other in the three-dimensional space by multidimensional scaling gives results consistent with the limited amount of independent evidence.     

The positioning of rye homologous chromosomes added to wheat through the cell cycle in somatic cells untreated and treated with colchicine

The arrangement of chromosome pairs 5RL and 7R added to the wild type and the ph1b mutant line of hexaploid wheat are analyzed in 2N somatic root tip cells during the cell cycle relative to the arrangement that chromosomes 5RL show in 4N tapetal cells produced after colchicine treatment. Both homologous chromosome pairs are identified at interphase and mitosis by fluorescence in situ hybridization. In nuclei at interphase, chromosomes appear as discrete domains that show the Rabl orientation. Homologous chromosomes are predominantly non-associated and their positioning seems not to be influenced by the Ph1 gene that suppresses homoeologous meiotic pairing. This pattern of arrangement contrasts with the high level of somatic pairing that sister chromosomes show in the interphase that follows chromosome duplication induced by colchicine. Disruption of pairing observed in some 4N nuclei is produced at c-anaphase which suggests no topological redistribution of homologues during conformation of the new nucleus. Homologous chromosomes show no predominant arrangement in ellipsoidal metaphase plates, which contrasts with the preferential opposite location of homologues in human prometaphase rosettes. Differences between chromosomes in the variation of the length through the cell cycle and in the chromatin morphology when the Ph1 is absent suggest different patterns of chromatin condensation in both chromosomes.     

Chromosome and DNA methylation dynamics during meiosis in the autotetraploid Arabidopsis arenosa

Variation in chromosome number due to polyploidy can seriously compromise meiotic stability. In autopolyploids, the presence of more than two homologous chromosomes may result in complex pairing patterns and subsequent anomalous chromosome segregation. In this context, chromocenter, centromeric, telomeric and ribosomal DNA locus topology and DNA methylation patterns were investigated in the natural autotetraploid, Arabidopsis arenosa. The data show that homologous chromosome recognition and association initiates at telomeric domains in premeiotic interphase, followed by quadrivalent pairing of ribosomal 45S RNA gene loci (known as NORs) at leptotene. On the other hand, centromeric regions at early leptotene show pairwise associations rather than associations in fours. These pairwise associations are maintained throughout prophase I, and therefore likely to be related to the diploid-like behavior of A. arenosa chromosomes at metaphase I, where only bivalents are observed. In anthers, both cells at somatic interphase as well as at premeiotic interphase show 5-methylcytosine (5-mC) dispersed throughout the nucleus, contrasting with a preferential co-localization with chromocenters observed in vegetative nuclei. These results show for the first time that nuclear distribution patterns of 5-mC are simultaneously reshuffled in meiocytes and anther somatic cells. During prophase I, 5-mC is detected in extended chromatin fibers and chromocenters but interestingly is excluded from the NORs what correlates with the pairing pattern.     

SCFSlimb ubiquitin ligase suppresses condensin II–mediated nuclear reorganization by degrading Cap-H2

Condensin complexes play vital roles in chromosome condensation during mitosis and meiosis. Condensin II uniquely localizes to chromatin throughout the cell cycle and, in addition to its mitotic duties, modulates chromosome organization and gene expression during interphase. Mitotic condensin activity is regulated by phosphorylation, but mechanisms that regulate condensin II during interphase are unclear. Here, we report that condensin II is inactivated when its subunit Cap-H2 is targeted for degradation by the SCF^Slimb ubiquitin ligase complex and that disruption of this process dramatically changed interphase chromatin organization. Inhibition of SCF^Slimb function reorganized interphase chromosomes into dense, compact domains and disrupted homologue pairing in both cultured Drosophila cells and in vivo, but these effects were rescued by condensin II inactivation. Furthermore, Cap-H2 stabilization distorted nuclear envelopes and dispersed Cid/CENP-A on interphase chromosomes. Therefore, SCF^Slimb-mediated down-regulation of condensin II is required to maintain proper organization and morphology of the interphase nucleus.     

Interphase chromosomes undergo constrained diffusional motion in living cells

Background: Structural studies of fixed cells have revealed that interphase chromosomes are highly organized into specific arrangements in the nucleus, and have led to a picture of the nucleus as a static structure with immobile chromosomes held in fixed positions, an impression apparently confirmed by recent photobleaching studies. Functional studies of chromosome behavior, however, suggest that many essential processes, such as recombination, require interphase chromosomes to move around within the nucleus.Results: To reconcile these contradictory views, we exploited methods for tagging specific chromosome sites in living cells of Saccharomyces cerevisiae with green fluorescent protein and in Drosophila melanogaster with fluorescently labeled topoisomerase ll. Combining these techniques with submicrometer single-particle tracking, we directly measured the motion of interphase chromatin, at high resolution and in three dimensions. We found that chromatin does indeed undergo significant diffusive motion within the nucleus, but this motion is constrained such that a given chromatin segment is free to move within only a limited subregion of the nucleus. Chromatin diffusion was found to be insensitive to metabolic inhibitors, suggesting that it results from classical Brownian motion rather than from active motility. Nocodazole greatly reduced chromatin confinement, suggesting a role for the cytoskeleton in the maintenance of nuclear architecture.Conclusions: We conclude that chromatin is free to undergo substantial Brownian motion, but that a given chromatin segment is confined to a subregion of the nucleus. This constrained diffusion is consistent with a highly defined nuclear architecture, but also allows enough motion for processes requiring chromosome motility to take place. These results lead to a model for the regulation of chromosome interactions by nuclear architecture.     

Specific staining of human chromosomes in Chinese hamster x man hybrid cell lines demonstrates interphase chromosome territories

Summary In spite of Carl Rabl's (1885) and Theodor Boveri's (1909) early hypothesis that chromosomes occupy discrete territories or domains within the interphase nucleus, evidence in favor pf this hypothesis has been limited and indirect so far in higher plants and animals. The alternative possibility that the chromatin fiber of single chromosomes might be extended throughout the major part of even the whole interphase nucleus has been considered for many years. In the latter case, chromosomes would only exist as discrete chromatin bodies during mitosis but not during interphase. Both possibilities are compatible with Boveri's well established paradigm of chromosome individuality. Here we show that an active human X chromosome contained as the only human chromosome in a Chinese hamster x man hybrid cell line can be visualized both in metaphse plates and in interphase nuclei after in situ hybridization with either ³H- or biotin-labeled human genomic DNA. We demonstrate that this chromosome is organized as a distinct chromatin body throughout interphase. In addition, evidence for the territorial organization of human chromosomes is also presented for another hybrid cell line containing several autosomes and the human X chromosome. These findings are discussed in the context of our present knowledge of the organization and topography of interphase chromosomes. General applications of a strategy aimed at specific staining of individual chromosomes in experimental and clinical cytogenetics are briefly considered.     

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.     

Nucleolar behaviour in Triticum

The maximum number of major nucleoli (macronucleoli) per nucleus of hexaploid, tetraploid and diploid wheat, Aegilops speltoides and Ae. squarrosa corresponded to the number of satellited chromosomes of each species. Smaller nucleoli (micronucleoli) were rare or absent in all of these species except the hexaploid, in which they were predominantly organized on chromosome arm 5D^s. — Fewer than the maximum number of macronucleoli in a mitotic interphase nucleus resulted from fusion of developing nucleoli. Enforced proximity of nucleolus-organizing regions resulted in more frequent fusion of nucleoli. — Analyses of related interphase nuclei showed that nucleoli, and hence probably chromosomes, undergo limited movement during mitotic interphase. These observations also indicate that specific chromosomes do not occupy specific sites in the interphase nucleus.     

SCHIP: statistics for chromosome interphase positioning based on interchange data

MOTIVATION: The position of chromosomes in the interphase nucleus is believed to be associated with a number of biological processes. Here, we present a web-based application that helps analyze the relative position of chromosomes during interphase in human cells, based on observed radiogenic chromosome aberrations. The inputs of the program are a table of yields of pairwise chromosome interchanges and a proposed chromosome geometric cluster. Each can either be uploaded or selected from provided datasets. The main outputs are P-values for the proposed chromosome clusters. SCHIP is designed to be used by a number of scientific communities interested in nuclear architecture, including cancer and cell biologists, radiation biologists and mathematical/computational biologists.     

Localization of DNA in the condensed interphase chromosomes of Euglena

The localization of DNA in the condensed interphase chromosomes of Euglena was determined by immunoelectron microscopy. Deposits of gold particles that coincided with the localization of DNA followed threads that corresponded to the chromatin fibers. The threads were 55–80 nm in diameter and were assumed to be supersolenoids. The localization of gold deposits on chromosomes that had been sectioned in various directions suggested that the chromatin fibers coiled around the surface of chromosomes, with a wide central axial region of the chromosomes remaining free of DNA. These findings are discussed in relation to current models of chromosomal structure.     

Studies in the Mitosis of Euglena I. On the Chromosome Cycle of Euglena viridis Ehrbg.

The chromosome cycle in the vegetative division of Euglena viridis was investigated. The seeming chromatin granules in the interphase nucleus are in reality thread structures, paired and very loosely twisted. Each component of the paired threads is called a chromatid, and consists of a fine thread of even thickness, the chromonema.
In the prophase, linear contraction and thickening of the chromatids occurs by means of the spiralization of them. In the later prophase, the coiled chromonema splits into two finer strands which show the plectonemic spiral. At the metaphase, the chromosomes are arranged in the form of an equatorial ring, encircling the median portion of the elongated endosome. Nearly all of the chromosomes have a submedian or a sub-terminal and a few of them have a terminal kinetochore. In the early anaphase, separation of the sister chromosomes takes place beginning at the kinetochore. The spindle fibres in the metaphase and anaphase were not observed. The two stranded spiral in the chromosomes is separated into distinct components by the uncoiling in the later telophase, and they are transformed, in the interphase nucleus, into the paired chromatids.     

Common pathway of chromosome condensation in Mammalian cells

Chromatin folding in the interphase nucleus is not known. We compared the pattern of chromatin condensation in Indian muntjac, Chinese hamster ovary, murine pre B, and K562 human erythroleukemia cells during the cell cycle. Fluorescent microscopy showed that chromosome condensation follows a general pathway. Synchronized cells were reversibly permeabilized and used to isolate interphase chromatin structures. Based on their structures two major categories of intermediates were distinguished: (1) decondensed chromatin and (2) condensed chromosomal forms. (1) Chromatin forms were found between the G1 and mid-S phase involving veil-like, supercoiled, fibrous, ribboned structures; (2) condensing chromosomal forms appeared in the late-S, G2, and M phase, including strings, chromatin bodies, elongated pre-chromosomes, pre-condensed chromosomes, and metaphase chromosomes. Results demonstrate that interphase chromosomes are clustered in domains; condensing interphase chromosomes are linearly arranged. Our results raise questions related to telomer sequences and to the chemical nature of chromosome connectivity.     

Chromosome Architecture and Genome Organization

How the same DNA sequences can function in the three-dimensional architecture of interphase nucleus, fold in the very compact structure of metaphase chromosomes and go precisely back to the original interphase architecture in the following cell cycle remains an unresolved question to this day. The strategy used to address this issue was to analyze the correlations between chromosome architecture and the compositional patterns of DNA sequences spanning a size range from a few hundreds to a few thousands Kilobases. This is a critical range that encompasses isochores, interphase chromatin domains and boundaries, and chromosomal bands. The solution rests on the following key points: 1) the transition from the looped domains and sub-domains of interphase chromatin to the 30-nm fiber loops of early prophase chromosomes goes through the unfolding into an extended chromatin structure (probably a 10-nm “beads-on-a-string” structure); 2) the architectural proteins of interphase chromatin, such as CTCF and cohesin sub-units, are retained in mitosis and are part of the discontinuous protein scaffold of mitotic chromosomes; 3) the conservation of the link between architectural proteins and their binding sites on DNA through the cell cycle explains the “mitotic memory” of interphase architecture and the reversibility of the interphase to mitosis process. The results presented here also lead to a general conclusion which concerns the existence of correlations between the isochore organization of the genome and the architecture of chromosomes from interphase to metaphase.     

Chromosome Banding and DNA Methylation Patterns, Chromatin Organisation and Nuclear DNA Content in Zingeria biebersteiniana

Chromosome structure and chromatin organisation of a two-chromosome model cereal Zingeria biebersteiniana (Claus) P. Smirnov were studied: nuclear DNA content was determined by microdensitometric analysis after Feulgen staining; Feulgen absorption at different thresholds of absorbance in interphase nuclei also provided evidence on the organisation of chromatin, allowing quantitative estimation of condensed chromatin within interphasic nucleus. The DNA methylation pattern of Z. biebersteiniana metaphase chromosomes was examined with a specific monoclonal antibody. 5-methyl-cytosine residues are present in several chromosome sites and differences may be present between corresponding regions of homologues. Chromosome banding pattern reveals large bands in the centromeric regions of each chromosome, showing constitutive heterochromatin; by fluorochromes staining pericentromeric blocks are evidenced. After the cold and 9-aminoacridine pre-treatments and after aceto-carmine and aceto-orceine staining, respectively, the metaphase chromosomes were analysed by image analysis system revealing a segmentation of the chromosome body that resembles Giemsa/Reverse banding in animal chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Ionizing radiation-induced instant pairing of heterochromatin of homologous chromosomes in human cells

Using fluorescence in situ hybridization with human band-specific DNA probes we examined the effect of ionizing radiation on the intra-nuclear localization of the heterochromatic region 9q12-->q13 and the euchromatic region 8p11.2 of similar sized chromosomes 9 and 8 respectively in confluent (G1) primary human fibroblasts. Microscopic analysis of the interphase nuclei revealed colocalization of the homologous heterochromatic regions from chromosome 9 in a proportion of cells directly after exposure to 4 Gy X-rays. The percentage of cells with paired chromosomes 9 gradually decreased to control levels during a period of one hour. No significant changes in localization were observed for chromosome 8. Using 2-D image analysis, radial and inter-homologue distances were measured for both chromosome bands. In unexposed cells, a random distribution of the chromosomes over the interphase nucleus was found. Directly after irradiation, the average inter-homologue distance decreased for chromosome 9 without alterations in radial distribution. The percentage of cells with inter-homologue distance <3 micro m increased from 11% in control cells to 25% in irradiated cells. In contrast, irradiation did not result in significant changes in the inter-homologue distance for chromosome 8. Colocalization of the heterochromatic regions of homologous chromosomes 9 was not observed in cells irradiated on ice. This observation, together with the time dependency of the colocalization, suggests an underlying active cellular process. The biological relevance of the observed homologous pairing remains unclear. It might be related to a homology dependent repair process of ionizing radiation induced DNA damage that is specific for heterochromatin. However, also other more general cellular responses to radiation-induced stress or change in chromatin organization might be responsible for the observed pairing of heterochromatic regions.     

Arrangement of chromatin in the nucleus

Summary The factors responsible for producing some degree of order to the arrangement of chromatin in the nucleus are reviewed. They are the following: 1. Chromosomes are attached to the nuclear membrane, nucleolus and intranuclear matrix. As a result they have a relatively fixed position in the nucleus. 2. In some species somatic pairing results in alignment of homologs. This is rare in mammals. 3. The association of ribosomal DNA and 5S DNA with the nucleolous results in the close approximation of the chromosomes carrying these DNA sequences. In man and other animals the most obvious consequence is satellite association. 4. Heterochromatin is condensed onto the inner nuclear membrane and periphery of the nucleolous while genetically active chromatin occupies the more central portion of the nucleus. The results is a peripheral location of late replicating DNA and a central location of early relicating DNA. 5. The DNA replication points tend to be associated with the nuclear matrix. Autoradiography of briefly labelled cells shows a high frequency of grains associated with nuclear matrix material. 6. Heterochromatin association results in chromocenters and ectopic pairing. 7. In addition to all these is the Rabl orientation or alignment of centromeres with centromeres and telomeres with telomeres. This polarization of the chromosomes results from the traction on the centromeres by the spindle fibers. There is no firm evidence for any higher degrees of order that might bring specific functioning genes into close proximity.Supported by NIH Grant GM 15886     

Do individual allocyclic chromosomes in metaphase reflect their interphase domains?

Summary Individual S phase allocyclic chromosomes have been analyzed in Bloom syndrome lymphocytes, in cells with an r(9), and in hypotetraploid Ehrlich mouse ascites cells treated with 1-methyl-2-benzyl hydrazine. On the basis of the following observations, we conclude that such chromosomes more or less reflect their domains in interphase: (1) The S phase allocyclic chromosomes have the same structure as S phase prematurely condensed chromatin (PCC) in fused cells; in other words they form limited areas of chromatin dots; (2) the allocyclic chromosome is the only chromosome in a metaphase plate which synthesizes DNA simultanneously with interphase nuclei; (3) the size of the allocyclic chromosomes is related to the size of the corresponding metaphase chromosome; and (4) the S phase allocyclic chromosomes resemble closely the chromosome domains in interphase made visible with biotinylated human DNA. A variety of evidence shows that most allocyclic chromosomes are simply left behind in their cycle, which presumably is caused by a deletion or inactivation of a hypothetical coiling center situated on each chromosome arm.