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      Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes

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          Abstract

          Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications during development and cell differentiation. However, the extent and cell-type-specificity of three-dimensional (3D) chromosome arrangements has remained controversial. In order to overcome technical limitations of previous studies, we have developed tools that allow the quantitative 3D positional mapping of all chromosomes simultaneously. We present unequivocal evidence for a probabilistic 3D order of prometaphase chromosomes, as well as of chromosome territories (CTs) in nuclei of quiescent (G0) and cycling (early S-phase) human diploid fibroblasts (46, XY). Radial distance measurements showed a probabilistic, highly nonrandom correlation with chromosome size: small chromosomes—independently of their gene density—were distributed significantly closer to the center of the nucleus or prometaphase rosette, while large chromosomes were located closer to the nuclear or rosette rim. This arrangement was independently confirmed in both human fibroblast and amniotic fluid cell nuclei. Notably, these cell types exhibit flat-ellipsoidal cell nuclei, in contrast to the spherical nuclei of lymphocytes and several other human cell types, for which we and others previously demonstrated gene-density-correlated radial 3D CT arrangements. Modeling of 3D CT arrangements suggests that cell-type-specific differences in radial CT arrangements are not solely due to geometrical constraints that result from nuclear shape differences. We also found gene-density-correlated arrangements of higher-order chromatin shared by all human cell types studied so far. Chromatin domains, which are gene-poor, form a layer beneath the nuclear envelope, while gene-dense chromatin is enriched in the nuclear interior. We discuss the possible functional implications of this finding.

          Abstract

          Through advanced microscopy and labeling techniques these authors can visualize and identify all of the chromosomes in a human nucleus--a key landmark towards understanding how our genome is regulated

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          Most cited references 62

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          Differences in the Localization and Morphology of Chromosomes in the Human Nucleus

          Using fluorescence in situ hybridization we show striking differences in nuclear position, chromosome morphology, and interactions with nuclear substructure for human chromosomes 18 and 19. Human chromosome 19 is shown to adopt a more internal position in the nucleus than chromosome 18 and to be more extensively associated with the nuclear matrix. The more peripheral localization of chromosome 18 is established early in the cell cycle and is maintained thereafter. We show that the preferential localization of chromosomes 18 and 19 in the nucleus is reflected in the orientation of translocation chromosomes in the nucleus. Lastly, we show that the inhibition of transcription can have gross, but reversible, effects on chromosome architecture. Our data demonstrate that the distribution of genomic sequences between chromosomes has implications for nuclear structure and we discuss our findings in relation to a model of the human nucleus that is functionally compartmentalized.
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            Dynamic organization of DNA replication in mammalian cell nuclei: spatially and temporally defined replication of chromosome-specific alpha-satellite DNA sequences

            Five distinct patterns of DNA replication have been identified during S- phase in asynchronous and synchronous cultures of mammalian cells by conventional fluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. During early S-phase, replicating DNA (as identified by 5-bromodeoxyuridine incorporation) appears to be distributed at sites throughout the nucleoplasm, excluding the nucleolus. In CHO cells, this pattern of replication peaks at 30 min into S-phase and is consistent with the localization of euchromatin. As S-phase continues, replication of euchromatin decreases and the peripheral regions of heterochromatin begin to replicate. This pattern of replication peaks at 2 h into S-phase. At 5 h, perinucleolar chromatin as well as peripheral areas of heterochromatin peak in replication. 7 h into S-phase interconnecting patches of electron-dense chromatin replicate. At the end of S-phase (9 h), replication occurs at a few large regions of electron-dense chromatin. Similar or identical patterns have been identified in a variety of mammalian cell types. The replication of specific chromosomal regions within the context of the BrdU-labeling patterns has been examined on an hourly basis in synchronized HeLa cells. Double labeling of DNA replication sites and chromosome-specific alpha-satellite DNA sequences indicates that the alpha-satellite DNA replicates during mid S-phase (characterized by the third pattern of replication) in a variety of human cell types. Our data demonstrates that specific DNA sequences replicate at spatially and temporally defined points during the cell cycle and supports a spatially dynamic model of DNA replication.
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              Tissue-specific spatial organization of genomes

              Background Chromosomes represent the largest structural units of eukaryotic genomes. The physically distinct nature of each chromosome is clearly visible during mitosis, when chromosomes condense and appear as separate entities. Chromosome-painting techniques have demonstrated that chromosomes are also physically separated during interphase, when each chromosome occupies a well defined nuclear subvolume, referred to as a chromosome territory [1,2]. The positioning of chromosomes during interphase is generally nonrandom [1,3,4]. In cells of plants with large genomes and of Drosophila melanogaster, centromeres and telomeres are positioned at opposite sides of the nucleus, giving rise to a chromosome arrangement known as the Rabl configuration [1,3,5]. In mammalian cells this pattern of genome organization is rare; instead, the spatial organization of chromosomes can be described by their radial positioning relative to the center of the nucleus [1,3,6]. In human lymphocytes, the radial positioning of chromosomes correlates with their gene density, with gene-dense chromosomes located towards the center of the nucleus and gene-poor chromosomes preferentially located towards the periphery [7,8]. Remarkably, the preferential radial positioning of at least two chromosomes, 18 and 19, has been evolutionarily conserved over 30 million years [9]. In addition to radial positioning, the nonrandom nature of genome organization is also reflected in the positioning of chromosomes relative to each other [10]. For example, in a lymphoma cell line derived from an ATM-/- mouse, two translocated chromosomes are preferentially positioned in close proximity to each other and the three chromosomes from which the translocations originated from a close-packed cluster in normal lymphocytes [10]. This type of nonrandom relative positioning has been proposed to facilitate formation of translocations by increasing the probability of illegitimate joining of broken chromosome ends of proximally positioned chromosomes [3,11,12]. While it is now well established that chromosomes are nonrandomly positioned [3,13,14], it is unclear how similar the spatial organization of the genome is in different tissues. Analysis of the radial positions of chromosomes 18 and 19 in different cell types failed to find significant differences [15]. Furthermore, a comparison of the distribution of several chromosomes in tissue-cultured fibroblasts and lymphoblasts gave mixed results: the position of several chromosomes appeared to be largely conserved between the two cell types, but on the other hand, chromosomes 6, 8, and 21 were positioned differently [7]. In both studies only radial positioning was used as a single indicator and distributions were not directly compared to each other by statistical means [7,15]. In an attempt to probe the spatial arrangement of chromosomes among tissues more systematically, we report here the comparative mapping of a subset of chromosomes in the cell nucleus of several cell types. From statistical analysis of several positioning criteria, including radial positioning, relative positioning, distance measurements and chromosome cluster analysis, we report evidence for tissue-specificity in the spatial organization of genomes. Results and discussion We sought to investigate the nuclear position of chromosomes 1, 5, 6, 12, 14 and 15 in a range of primary cells freshly isolated from mouse tissues. We visualized single chromosomes by fluorescence in situ hybridization (FISH) using chromosome-specific probes and analyzed their position in normal interphase cells containing a diploid complement of fluorescent signals (Figure 1). Freshly isolated and minimally cultured primary cell populations were used to prevent potential reorganization of chromosomes during prolonged in vitro culture. Qualitative inspection of the distribution of painted chromosomes indicated tissue specificity in chromosome positioning (Figure 1a). For example, chromosome 5 was preferentially found towards the center of the nucleus in liver cells, was predominantly peripheral in small and large lung cells, but was located in an intermediate position in lymphocytes (Figure 1a). For quantitative analysis of positioning, we first measured the distance between the nuclear center and the center of mass of each chromosome signal as an indicator of its radial position in two-dimensional (2D) projections of three-dimensional (3D) image stacks as previously described (Figure 1b; see also Materials and methods) [8,11]. The distribution profiles of chromosomes showed considerable differences among tissues (Figure 1b). Statistical analysis of pairwise comparisons of the distribution of single chromosomes using contingency table analysis among all tissues revealed highly significant differential radial positioning (Figure 1c). Differential positioning in at least three cell types was found for all chromosomes analyzed (Figure 1b,c). Out of 71 pairwise comparisons, 34 were statistically significant at the p 0.5), but not of chromosomes 5, 6 and 15 (Figure 1b,c; all p-values 73.91% nuclear radius. Bins defined in this manner represent volumes with equal probability of containing the same number of chromosomes assuming a uniform random distribution of 40 spherical chromosomes of excluding volume with a radius of 10% of the nuclear volume in a spherical nucleus. To test for differences in average minimum separation of chromosome pairs between cell types we applied the Kolmogorov-Smirnov test [36]. To test for differences in proximal triplet formation, we constructed contingency tables for the frequencies of the experimentally observed four categories of chromosome arrangements [34,35]. Triplets were defined as a collection of three chromosome pairs all separated by less than 30% of nuclear diameter [10]. The contingency table analysis tested the null hypothesis that all four categories of chromosome arrangements are equally likely and independent of the cell type. To determine tissue-specific proximity of translocation partners we determined the frequencies of cells containing at least one close pair of either 5-6 or 12-15 in lymphocytes and hepatocytes as previously described [11]. We defined a close pair as two chromosomes located at a distance not larger that 20% of the nuclear diameter. Frequencies of pair formation were analyzed analogously to the triplet analysis using the null hypothesis that the number of close pairs in a given cell type is independent of the identities of the chromosomes. All analyses were done using standard algorithms coded in Java. For all experiments data from at least three independent experiments was pooled. Additional data files The following additional files are available with the online version of this paper: contingency tables for chromosomes 12, 14, and 15 triplet formation (Additional data file 1); chromosomes 1, 12 and 14 triplet formation (Additional data file 2); chromosomes 1, 12 and 15 triplet formation (Additional data file 3); and chromosomes 1, 14 and 15 triplet formation (Additional data file 4). Supplementary Material Additional data file 1 Contingency table for chromosomes 12, 14, and 15 triplet formation Click here for additional data file Additional data file 2 Contingency table for chromosomes 1, 12 and 14 triplet formation Click here for additional data file Additional data file 3 Contingency table for chromosomes 1, 12 and 15 triplet formation Click here for additional data file Additional data file 4 Contingency table for chromosomes 1, 14 and 15 triplet formation Click here for additional data file
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS Biol
                pbio
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                1544-9173
                1545-7885
                May 2005
                26 April 2005
                : 3
                : 5
                Affiliations
                1Department of Biology II, Anthropology and Human Genetics Ludwig Maximilians University, MunichGermany
                2Kirchhoff Institute of Physics, University of Heidelberg HeidelbergGermany
                3Theoretical Bioinformatics, German Cancer Research Center (DKFZ) HeidelbergGermany
                4Institute of Human Genetics, Technical University Munich Germany
                5Institute of Human Genetics, GSF National Research Center for Environment and Health NeuherbergGermany
                National Cancer Institute United States of America
                Article
                10.1371/journal.pbio.0030157
                1084335
                15839726
                e0d03f96-5a42-4e11-9bd3-955965e56af6
                Copyright: © 2005 Bolzer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited
                Categories
                Research Article
                Cell Biology
                Genetics/Genomics/Gene Therapy
                Homo (Human)

                Life sciences

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