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
