Journal of Cell Science 112, 525-535 (1999) 525 Printed in Great Britain © The Company of Biologists Limited 1999 JCS4591 Chromosomes exhibit preferential positioning in nuclei of quiescent human cells Robert G. Nagele1,*, Theresa Freeman1, Lydia McMorrow2, Zabrina Thomson3, Kelly Kitson-Wind1 and Hsin-yi Lee3 1Department of Molecular Biology, University of Medicine and Dentistry of New Jersey/SOM, 2 Medical Center Drive, Stratford, New Jersey 08084, USA 2Department of Laboratory Science, Thomas Jefferson University, Philadelphia, Pennsylvania, 19107, USA 3Department of Biology, Rutgers University, Camden, New Jersey 08102, USA *Author for correspondence (e-mail: [email protected]) Accepted 20 November 1998; published on WWW 25 January 1999 SUMMARY The relative spatial positioning of chromosomes 7, 8, 16, X predictable within remarkably narrow spatial limits. Dual- and Y was examined in nuclei of quiescent (noncycling) FISH with various combinations of chromosome-specific diploid and triploid human fibroblasts using fluorescence DNA probes and contrasting fluorochromes was used to in situ hybridization (FISH) with chromosome-specific identify adjacent chromosomes in mitotic rosettes and test DNA probes and digital imaging. In quiescent diploid cells, whether they are similarly positioned in interphase nuclei. interhomolog distances and chromosome homolog position From among the combinations tested, chromosomes 8 and maps revealed a nonrandom, preferential topology for 11 were found to be closely apposed in most mitotic rosettes chromosomes 7, 8 and 16, whereas chromosome X and interphase nuclei. Overall, results suggest the existence approximated a more random distribution. Variations in of an ordered interphase chromosome topology in the orientation of nuclei on the culture substratum tended quiescent human cells in which at least some chromosome to hinder detection of an ordered chromosome topology at homologs exhibit a preferred relative intranuclear location interphase by biasing homolog position maps towards that may correspond to the observed spatial order of random distributions. Using two chromosome X homologs chromosomes in rosettes of mitotic cells. as reference points in triploid cells (karyotype = 69, XXY), the intranuclear location of chromosome Y was found to be Key words: Mitosis, Chromosome, Chromosome topology, Human INTRODUCTION chromosome position in interphase nuclei would imply that the nuclear context in which genes operate is critical for their Our understanding of the structure and organization of the normal expression as originally suggested by Blobel (1985). interphase nucleus have recently undergone a major revision Thus far, the results of studies on chromosome topology in due to parallel advancements including refinements in the interphase nuclei have been contradictory. A number of studies fluorescence in situ hybridization (FISH) technique, the on different cell types have shown that the intranuclear locations development of a wide variety of chromosome- and gene- of at least some chromosome territories are nonrandom and may specific DNA probes, and dramatic advances in light be both cell type- and cell cycle-specific (for reviews, see microscopy and digital imaging. For example, a number of Heslop-Harrison and Bennett, 1984; Manuelidis, 1990; Haaf and studies using FISH with chromosome painting probes have Schmid, 1991; Spector, 1993). For example, in human and shown that chromosomes occupy relatively compact, non- mouse central nervous system cells, some chromosome domains overlapping territories in interphase nuclei of both animal and exhibit similar distribution patterns (Manuelidis and Borden, plant cells (Cremer et al., 1982, 1988; Lichter et al., 1988; 1988). In human fibroblasts, the distribution of chromosome 8 Hilliker and Appels, 1989; Schwarzacher et al., 1989; Leitch centromeric regions is nonrandom and changes during the cell et al., 1990; Manuelidis, 1990; Haaf and Schmid, 1991; cycle (Popp et al., 1990; Ferguson and Ward, 1992). Spector, 1993). The question as to whether or not these Chromosome 1 centromeric DNA localizes selectively to the chromosome territories indeed exhibit a relative spatial order nuclear periphery in hemopoietic cells (Van Dekken et al., 1989). within interphase nuclei has been hotly debated over many Likewise, the inactivated X chromosome maintains a peripheral years (Comings, 1968, 1980; Heslop-Harrison and Bennett, distribution in interphase nuclei of fibroblasts (Manuelidis and 1984; Spector, 1993). The biological significance of this issue Borden, 1988). In addition, distribution patterns of centromeres cannot be overrated, particularly because control over and telomeres vary with cell type and cell cycle phase, and may 526 R. G. Nagele and others be influenced by the state of cell differentiation (Ferguson and Cell Repository). Cells were grown on glass coverslips in Dulbecco’s Ward, 1992; Manuelidis, 1985; Hadlaczky et al., 1986; Haaf and Modified Eagle Medium (DMEM) (Gibco, BRL) containing 10% fetal Schmid, 1989; Bartholdi, 1991; Vourc’h et al., 1993). Active calf serum (Gibco, BRL) and 1% penicillin/streptomycin at 37°C in a genes are also distributed nonrandomly in nuclei, concentrating 5% CO2 atmosphere. Some cultures were grown to a state of ‘high- in the nuclear periphery of mouse L and P19 embryonal density confluence’ and maintained in this condition for 2-4 days carcinoma cells and at the edges of condensed chromatin in newt without medium change. Cell cycle analysis (see below) showed that this procedure ‘locked’ cells in the G1/G0 phase of the cell cycle and erythrocytes (Hutchison and Weintraub, 1985). On the other effectively minimized individual cell and nuclear shape variations that hand, a number of other studies have failed to detect distinct would otherwise have arisen as a result of such complex behaviors as chromosome distribution patterns in nuclei, and thus support a cell migration, cell spreading, and mitosis. Confirmation of cell cycle random intranuclear distribution of chromosomes at interphase. arrest was obtained by DNA quantitation, preparation of cell cycle For example, Vourc’h et al. (1993) have shown that chromatin analysis profiles as described below, and examination of cultures for the is redistributed in the interphase nucleus of mouse lymphocytes occurrence of mitotic figures. during the cell cycle. Similarly, Lesko et al. (1995) demonstrated that the relative distributions of chromosomes 7, 11 and 17 in Cell cycle analysis interphase T lymphocytes could not be distinguished from those High-density confluent cell cultures were analyzed using the Cell obtained from random points in a truncated sphere. This Analysis System 200 (CAS 200, Beckman). The CAS 200 uses a stoichiometric Feulgen staining reaction and microscopic observation is in agreement with the data of Ferguson and Ward densitometry to determine the total DNA content of individual nuclei (1992), who measured the distances and angles between and generates cell cycle profiles for cultures (Fig. 1). The main homologs in flow-sorted T lymphocyte nuclei and reported that advantages of the CAS 200 over fluorescence cell sorters is that the the arrangement of chromosome homologs relative to each other analysis can be done on cells already affixed to microslides and a is not spatially defined. Arnoldus et al. (1989) reported the single nucleus can be analyzed by FISH. Based on the total amount interphase association of the two 1q12 heterochromatin regions of nuclear DNA, each nucleus can be assigned a specific phase of the in nuclei from the human cerebellum, but were unable to cell cycle. In the present study, the CAS 200 was also used to confirm demonstrate this association in the cerebral cortex. that cells in high-density confluent cultures were ‘locked’ in the G0/G1 Heterochromatin of the active X chromosome in cultured human phase. Data are presented as a histogram which shows the relative cells has been reported to be randomly distributed (Popp et al., numbers of cells in the G0/G1, S, and G2/M phases. For individual nuclei, results are expressed as picograms of DNA per nucleus. The 1990). Taken together, the existence of an organized replication precision of measurements of individual nuclei and the chromosome topology at interphase is still a matter for debate, stability of calibration were tested by comparing measurements of 25 and a common organizing principle that influences or dictates consecutive images of the same nucleus. interphase chromosome topology remains elusive. In the present study, we have investigated the topology of Fluorescence in situ hybridization (FISH) and digital chromosomes 7, 8, 16, X and Y in quiescent human diploid imaging and triploid fibroblasts using FISH and digital imaging. Cells grown on glass coverslips were fixed in 4% paraformaldehyde in Quiescent cells were chosen in an effort to minimize potential phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl, 16 mM variations in chromosome topology stemming from differences Na2HPO4, 2 mM KH2PO4, pH 7.3) for 20 minutes at room temperature. in cell type, cell cycle phase, cell and nuclear shape and Fixed specimens were washed briefly in PBS, followed by three rinses in 2× SSC (1× SSC; 150 mM NaCl, 30 mM sodium citrate) for 10 physiological state. They also provide an excellent model minutes at room temperature. For digoxigenin-labeled, whole- system because cells and their nuclei are flattened against the chromosome painting probes, cellular DNA was denatured at 70°C for culture substratum during