The three-dimensional organization of telomeres in the nucleus of mammalian cells
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The observation of multiple genetic markers in situ by optical microscopy and their relevance to the study of three-dimensional (3D) chromosomal organization in the nucleus have been greatly developed in the last decade. These methods are important in cancer research because cancer is characterized by multiple alterations that affect the modulation of gene expression and the stability of the genome. It is, therefore, essential to analyze the 3D genome organization of the interphase nucleus in both normal and cancer cells.
We describe a novel approach to study the distribution of all telomeres inside the nucleus of mammalian cells throughout the cell cycle. It is based on 3D telomere fluorescence in situ hybridization followed by quantitative analysis that determines the telomeres' distribution in the nucleus throughout the cell cycle. This method enables us to determine, for the first time, that telomere organization is cell-cycle dependent, with assembly of telomeres into a telomeric disk in the G2 phase. In tumor cells, the 3D telomere organization is distorted and aggregates are formed.
The results emphasize a non-random and dynamic 3D nuclear telomeric organization and its importance to genomic stability. Based on our findings, it appears possible to examine telomeric aggregates suggestive of genomic instability in individual interphase nuclei and tissues without the need to examine metaphases. Such new avenues of monitoring genomic instability could potentially impact on cancer biology, genetics, diagnostic innovations and surveillance of treatment response in medicine.
KeywordsInterphase Nucleus Burkitt Lymphoma Cell Line Normal Human Epithelial Tissue Mouse Fetal Liver Cell Significant Directional Movement
Cancer is characterized by multiple alterations that affect the modulation of gene expression and the stability of the genome. These interconnected changes occur within the nuclei of cells that alter their three dimensional (3D) organization during tumor initiation and progression [1, 2]. It seems reasonable to assume that the highly organized mammalian interphase nucleus is the structure that ascertains genomic stability. In line with these concepts, oncogenic activation remodels this nuclear order and sets the stage for genomic instability as we have recently measured for conditional c-Myc deregulation. The deregulated expression of c-Myc alters the 3D nuclear space of chromosomes and telomeres, and makes genomic rearrangements topologically feasible (Chuang et al., in preparation).
Defining the structural organization of the interphase nucleus is, therefore, essential to our understanding of the 3D genome organization in the interphase nucleus. Such a study can be performed by fluorescence in situ hybridization (FISH). Two of the most attractive features of FISH measurements of the 3D nucleus organization are the ability to simultaneously visualize multiple targets and the structural organization of nucleus and cells, something that cannot be achieved by array-based methods.
The organization of the interphase nucleus has been studied since the late nineteenth century . It is now well accepted that the position of chromosomes in the nucleus plays an important role in gene regulation . Nevertheless, some controversy exists. Most laboratories have observed a non-random organization of chromosome territories [2, 5, 6] that has been conserved during evolution . This has been further supported by studies that demonstrate an architectural stability of the chromosomal positions in the nucleus [8, 9]. There are, however, different observations on chromosomal positions [10, 11, 12, 13, 14, 15] as well as on positional changes of chromosomes during the cell cycle [16, 17].
Recently, interest has also focused on telomeres, whose importance to genomic stability was recognized as early as the 1930s . Capping the chromosomes, telomeres are responsible for chromosomal integrity  to prevent genomic instability . Some reports have been published on the 3D organization of telomeres in the nucleus, mainly with regard to the distances of telomeres from the nuclear shell. Telomeres have been previously found at the nuclear edge , at the nuclear periphery , throughout the entire nucleus [13, 23], in non-Rabl association , in association with the nucleolus  or in the nuclear matrix .
Telomere dynamics also have been studied in living human U2OS osteosarcoma cells . Individual telomeres showed significant directional movements and telomeres were shown to associate with promyelocytic leukemia bodies in a dynamic manner. This means that telomere structure is dynamic, and may be important for both transcriptional processes and for stabilizing chromosome positions in the nucleus.
We have developed a method of studying the organization of the genome by analysis of the 3D organization of telomeres in the nucleus and their positional changes along the cell cycle, using flow-sorted living cells. This method enables us to determine, for the first time, that telomere organization is cell-cycle dependent, with assembly of telomeres into a telomeric disk in the G2 phase. Moreover, we show for tumor cells that the 3D telomere organization is distorted and that telomeric aggregates are formed. These results emphasize a non-random and dynamic 3D nuclear telomeric organization and its importance to genomic stability.
Results and discussion
We first described the major observation of primary BALB/c mouse B lymphocytes that were studied along the cell cycle. These studies were followed by the analysis of immortalized cells. The lymphocytes were sorted according to their DNA content for the determination of the G0/G1, S or G2/M phases (see Methods).
By analyzing cell-cycle sorted primary mouse lymphocytes we found that the 3D telomere organization changes during the cell cycle. Telomeres are widely distributed throughout the nucleus in the G0/G1 and S phases with a calculated a/c ratio of 0.9 ± 0.4, which means a spherical-like volume of distribution. However, during G2, telomeres are not observed throughout the whole nucleus. Their 3D organization changes, with all the telomeres assuming a central structure that we call the telomeric disk, which has never been reported before. In this ordered structure, all the telomeres align in the centre of the nucleus as cells progress into the late G2 phase. The a/c ratio they assume is 6.0 ± 2.0, which means a very flat disk (almost a coin shape).
Similar results have been observed in primary human lymphocytes, primary human fibroblasts and in normal human epithelial tissue (see additional file for more data). This suggests that chromosomes assume a very precise order that pre-aligns them prior to the onset of mitosis. In order to ascertain that the telomeric disk was not the result of a distorted nucleus, our analysis programme compared the telomere distribution volume and shape with that of the 4'-6-Diamidino-2-phenylindole (DAPI) – stained nucleus, and verified that the nucleus itself still had a spherical-like volume. We rarely found distorted nuclei and excluded these cells from the analysis. The nucleus shown in G2 is not fully spherical. Such a shape is expected, because when the telomeres forms a disk, it pools the chromosomes and forces them to be closer to the disk, which results in an oblate shape as well.
These results reveal that the telomeric disk is formed in the late G2 phase. As cells progress from G2 to M, chromosomes organize into metaphases and, therefore, the number of cells in interphase with a telomeric disk decreases. Because there is no other state of transition between telomeric disk and mitosis, we conclude that the telomeric disk is the 3D telomeric organization assumed in late G2. Thus, it is also the final stage of the interphase nucleus that permits the organization of the genetic material prior to its entry into the M phase and prior to chromosome segregation. Cells in late G2 with a telomeric disk have additional characteristic features: i) they exhibit a larger overall nuclear volume than their G1 or S phase counterparts (this increase in size was also confirmed by fluorescent activated cell sorter [FACS] analysis); and ii) they begin to show signs of early re-organization of the chromatin into partially condensed areas (as visualized using the DAPI stained image).
At the end of the M phase, we observe cells that enter into the G1 conformation of telomeres, with a wide spatial distribution of telomeres throughout a smaller nucleus.
In conclusion, this data indicates that the telomeric disk is a novel structure within the interphase nucleus in late G2 that has not been previously described. Its existence points to the fundamental importance of ordered nuclear organization at the end of G2. The telomeric disk probably assures the proper organization of chromosomes prior to mitosis and their organized segregation during mitosis. Together with information that has been previously published on telomeric dynamics [26, 28], it is tempting to speculate that telomeres take an active part in the process of chromosome organization into a unique structure, the telomeric disk, during G2. This alignment of telomeres and chromosomes would facilitate the proper subsequent organization of the chromosomes into an equatorial plane during cell division. This process may be driven by the telomeres themselves (that are free of the nuclear matrix) or through the nuclear matrix. The telomeric disk may also allow for a late G2 checkpoint.
Further work on the subject can also be performed in vivo, as has been shown by Molenaar et al. . In such a way the full dynamic process can be observed, which is complementary to the single time-points that are shown in our work.
In line with these concepts, oncogenic activation remodels this nuclear order and sets the stage for genomic instability as we have recently measured for conditional c-Myc deregulation. We have found that deregulated expression of c-Myc alters the 3D nuclear organization of chromosomes and telomeres, and makes genomic rearrangements topologically feasible (Chuang et al., in preparation).
In summary, we have shown that 3D optical imaging followed by the analysis of telomeres in the interphase is an important tool for basic research and cancer biology. We have found cell-cycle dependence of the telomere organization in the nucleus, where telomeres align into a telomeric disk during the late G2 phase. Such an organization has never before been reported.
Telomeric aggregates are found in tumor cells and, therefore, an alteration of the telomeric disk is seen. Transient telomeric aggregations potentially cause irreversible chromosomal rearrangements.
The above findings indicate that it is now possible to examine the presence of telomeric aggregates suggestive of genomic instability in individual interphase nuclei and tissue, without the need to examine metaphases. Such new directions of monitoring genomic instability could potentially have an impact on cancer biology, genetics, diagnostic innovations and surveillance of treatment response in medicine.
Mouse primary cells were directly isolated from BALB/c mice and stimulated with lipopolysaccharide to enter into the cell cycle . Primary mouse fetal liver cells were also directly isolated from BALB/c mice. Mice were studied according to the protocols approved by Canadian Central Animal Care. Immortalized mouse pro B lymphocytes have been described elsewhere . Human primary cells were obtained from healthy donors. Head and neck squamous cell carcinoma and control tissue were obtained from a patient at CancerCare Manitoba upon ethics approval and informed consent.
Pro B lymphocytes  were fixed in four ways: i) following cytospin preparations, cells were fixed in 3.7% formaldehyde (1×PBS/50 mM MgCl2); ii) cells were allowed to grow on glass slides and were fixed in 1% formaldehyde (3D fixation); iii) cells were fixed in suspension with 3.7% formaldehyde (3D fixation); and iv) cells were fixed in methanol:acetic acid (3:1) according to standard protocols . Tissue was fixed following cryosection (5 μm sections were used) in 1% formaldehyde (1×PBS/50 mM MgCl2). All hybridizations shown in this report were carried out after 3D fixation.
Fluorescent activated cell sorter (FACS) analysis
For FACS analysis, primary lymphocytes were fixed in 70% cold ethanol and stained with propidium iodide (1 μg/mL) following RNAse (20 μg/mL) digestion. The stained cells were analysed for DNA content by flow cytometry in a EPICS Altra cytometer (Beckman-Coulter). Cell cycle fractions were quantified with WinCycle software (Phoenix Flow Systems, San Diego, CA).
Cells were stained with Hoechst 33342 (Molecular Probes) at a final concentration of 1 μg/mL for 90 minutes at 37°C and 5% of carbon dioxide (CO2). Cells were sorted according to their DNA content (G0/G1, S and G2/M phases) with a EPICS Altra cytometer (Beckman-Coulter) equipped with a UV laser (Coherent, excitation at 350 nm) and a 460 nm band-pass filter.
Pro B lymphocytes were labelled in vivo with 10 μM of BrdU (5-Bromo-2'-deoxyuridine, SIGMA-ALDRICHT, Lyon, France) for one hour at 37°C in humidified atmosphere (5% CO2). BrdU was then detected with 5 μL/1×106 cells of anti-BrdU-FITC (fluorescein isothiocyanate) antibody (TEBU, Le Perray-en-Yvelines, France) at identical conditions for 30 minutes. Thereafter, all BrdU (i.e. FITC)-positive cells were live sorted, placed into culture for different times and harvested at 3.5, 4, 5, 6, 7, 8, 8.5, 9 and 10 hours after labeling and sorting. The cells were then fixed for 3D analysis. For each time point we have measured: 1. the fraction of nuclei with a telomeric disk; 2. the fraction of cells in mitosis; and 3. the fraction of cells in interphase nuclei without telomeric disk and mitotic figures that were evaluated as G1 and S phase cells.
Telomere FISH using Cy3-labled PNA probes
Telomere FISH was performed as described  using a Cy3-labelled PNA probe (DAKO, Glostrup, Denmark). Telomere hybridizations were specific as shown by metaphase hybridizations and the correct number of the telomeric signals observed at the ends of chromosomes prepared from primary cells (Fig. 4).
3D image acquisition
Unless stated otherwise, 20–30 cells were analyzed by 3D imaging from each cell type and phase type. Part of the measurements were done with a confocal microscope (Leica AOBS-SP) and most of them with a conventional Axioplan 2 (Zeiss) with a cooled AxioCam HR CCD followed by deconvolution . DAPI, FITC and Cy3 filters (Zeiss) were used in combination with Planapo 63×/1.4 oil (Zeiss). Axiovision 3.1 software with a deconvolution module and rendering module were used (Zeiss). Both methods gave similar results.
80–100 sections were acquired for each 3D nucleus, typically with 200 × 200 pixels per section with a ~100 × 100 nm nominal imaging area per pixel (steps of 200 nm along Z). The point-spread function of our system has a full width at half max of approximately 200 nm in the plane and 400 nm along the optical axis.
3D analysis of telomeres
In order to analyze the telomere distribution in the nucleus, we developed a special 3D image analysis programme. The main algorithmic part is described below. The programme (TeloView) is based on the Matlab computer language (The MathWorks, Natick, MA, USA) and some of the image processing algorithms are based on the DipImage library (developed at the Quantitative Imaging Group, Delft University of Technology, Delft, The Netherlands) .
The programme segments the nucleus volume by a derivative-based algorithm using a morphological top and bottom-hat algorithm . The volume, intensity and centre of gravity are calculated for each spot. The programme then finds a principle plane in the nucleus (x'y') that is the closest to all the telomeres (Fig. 2). This is especially important when a tissue section is analyzed, because this plane should not necessarily be parallel to the microscope slide plane.
The telomeric distribution inside the nucleus is described by fitting an ellipsoid to the volume occupied by the telomeres (three different main axes; Fig. 2). The distributions were found to be either oblate or spherical (i.e. the two principle axes along the main x'y' plane of the spheroid are similar). It is, therefore, convenient to describe the distribution volume as a spheroid (i.e. an ellipsoid having two axes of equal length). As such, it is simpler to describe the spheroid degree of variation from a perfect sphere by the ratio a/c where a and b are the similar semi-axes and c is the third one. Such a description reflects the degree to which the telomere's volume is oblate.
The authors wish to thank our colleagues and lab members for discussions and critical reading of this manuscript. This work was supported by the Canada Foundation for Innovation, the Canadian Institutes of Health Research (NSERC), CancerCare Manitoba, Fondation de France (Paris), the French Minister of Foreign Affairs, Sander-Stiftung and Verein zur Förderung der Krebsforschung e.V.
This work was also supported by the Physics for Technology programme of the Foundation for Fundamental Research in Matter (FOM), the Delft Inter-Faculty Research Center Life Tech and the Delft Research programme Life Science and Technology.
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