Chromosome Research 11: 485^502, 2003. # 2003 Kluwer Academic Publishers. Printed in the Netherlands

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Three-dimensional arrangements of centromeres and telomeres in nuclei of human and murine lymphocytes

Claudia Weierich1 , Alessandro Brero1 , Stefan Stein2 , Johann von Hase2 , Christoph Cremer2 , Thomas Cremer1 & Irina Solovei1
1 Department of Biology II, Human Genetics, Ludwig Maximillians University (LMU), Richard Wagner Str. 10, 80333 Munich, Germany; Tel: þ 49-89-2180-6713; Fax: þ 49-89-2180-6719; E-mail: [email protected]; 2 Kirchhoff Institute of Physics, University of Heidelberg, Heidelberg, 69120 Germany *Correspondence

Key words: centromere, chromosome, confocal microscopy, £uorescence in-situ hybridization, interphase, lymphocyte, nuclear architecture, telomere

Abstract The location of centromeres and telomeres was studied in human and mouse lymphocyte nuclei (G0) employing 3D-FISH, confocal microscopy, and quantitative image analysis. In both human and murine lymphocytes, most centromeres were found in clusters at the nuclear periphery. The distribution of telomere clusters, however, differed: in mouse nuclei, most clusters were detected at the nuclear periphery, while, in human nuclei, most clusters were located in the nuclear interior. In human cell nuclei we further studied the nuclear location of individual centromeres and their respective chromosome territories (CTs) for chromosomes 1, 11, 12, 15, 17, 18, 20, and X. We found a peripheral location of both centromeres and CTs for 1, 11, 12, 18, X. A mostly interior nuclear location was observed for CTs 17 and 20 and the CTs of the NOR-bearing acrocentric 15 but the corresponding centromeres were still positioned in the nuclear periphery. Autosomal centromeres, as well as the centromere of the active X, were typically located at the periphery of the respective CTs. In contrast, in about half of the inactive X-CTs, the centromere was located in the territory interior. While the centromere of the active X often participated in the formation of centromere clusters, such a participation was never observed for the centromere of the inactive X.

Introduction Chromosomes occupy mutually exclusive nuclear domains in mammalian cell nuclei, called chromosome territories (CTs). Chromosome arm domains, as well as G- and R-band domains, occupy distinct subregions within these CTs with little chromatin intermingling (Dietzel et al. 1998a, 1998b, Zink et al. 1999, Chevret et al.

2000, Cremer & Cremer 2001). The radial nuclear arrangements of CTs and chromosomal subregions of di¡erent cell types and di¡erent species are non-random and evolutionary conserved suggesting that they may be important for nuclear functions (Cremer et al. 2000, Cremer & Cremer 2001, Dundr & Misteli 2001, Parada & Misteli 2002, Tanabe et al. 2002a). Knowledge of 3D locations of subchromosomal regions, however,

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486 has remained very limited to date. Some reports described evidence that actively transcribed sequences are situated at, or close to, the surface of interphase CTs, while silent genes and nontranscribed sequences are predominantly found in the inner portions of CTs (Kurz et al. 1996, Dietzel et al. 1999). It was also shown that the MHC locus is located at the periphery of the human CT #6 and that induction of the expression of MHC class II genes causes the expansion of large loops outwards from the CT periphery (Volpi et al. 2000). Other reports, however, indicate that transcription can also be observed in the CT interior (Abranches et al. 1998, Mahy et al. 2002a, 2002b). In order to explain these cases one should take into account the folding of CTs. This folding could result in invaginations of the CT surface allowing the direct contact of chromatin loop domains harboring the respective genes with the interchromatin compartment (Cremer & Cremer, 2001). It was recently shown that the association of genes with heterochromatic chromocenters contributes to their inactivation (Brown et al. 1997, 1999, Schubeler et al. 2000, Baxter et al. 2002). This ¢nding suggests that the topology of a centromere with regard to its respective territory and to the nucleus at large may play a role in the epigenetic mechanisms that control gene silencing. While a number of publications exist which describe the nuclear locations of centromeres (see below), their possible locations with respect to their CTs have not been analyzed in detail except for two recent publications: one reported that the centromere of human chromosome #6 occupies a peripheral CT position (Chevret et al. 2000); the other that the centromere of human chromosome #15 is mostly located in the interior of the CT (Nogami et al. 2000). Nuclear positions of centromeres and telomeres have been intensely studied. A tendency to a peripheral location of centromeres at G1/G0 was noted for both human (Ferguson & Ward 1992, Weimer et al. 1992, Alcobia et al. 2000, Skalnikova et al. 2000) and mouse cells (Vourc’h et al. 1993). Recent publications have focused on the arrangements of CTs in the interphase nucleus (reviewed by Parada & Misteli, 2002). It has been shown that CTs have a non-random radial dis-

tribution in spherical nuclei of lymphocytes and lymphoblastoid cells (Croft et al. 1999, Boyle et al. 2001, Cremer et al. 2001, Tanabe et al. 2002a, Tanabe et al. 2002b). CTs with a high gene density are located more centrally than gene-poor CTs. The question of how the internal position of genepoor CTs is compatible with the peripheral location of centromeres in spherical nuclei has not been resolved and requires the analysis of speci¢c centromere localizations in 3D preserved nuclei (Solovei et al. 2002b). The present study was focused on the spatial arrangements of speci¢c centromeres, including the centromeres of human chromosomes 1, 4, 11, 12, 13 þ 21, 14 þ 22, 15, 17, 18, 20 and respective chromosome territories (for most of them) in human non-stimulated (G0) lymphocytes from peripheral blood. Further, we wished to compare the centromere locations in active and inactive X-CTs. Since we were not able to identify the Barr body (X inactive) in lymphocyte nuclei, for this purpose we chose human female ¢broblasts where the Barr body could easily be identi¢ed. For comparison, the spatial arrangements of all centromeres, as well as all telomeres, were analyzed in nuclei from human and mouse G0 lymphocytes. To approach these matters we used two-color 3D-FISH and nucleoli immunostaining in combination with confocal microscopy. For the analysis of the distribution of £uorescently labeled DNA in spherical nuclei a specially designed computer program was used.

Material and methods Cells and ¢xation for FISH Human and mouse (hybrids C3HeB/FeJ) lymphocytes (G0) from peripheral blood were isolated in a Ficoll gradient and resuspended in Hanks’ balanced salt solution to a concentration of 1  106 cells/ml. Then 300-ml aliquots of this suspension were placed on coverslips coated with polylysine (1 mg/ml, Sigma) and cells were allowed to attach for 30^60 min at 37 C. Human skin ¢broblasts were grown on coverslips till con£uence (when almost all cells are in G0) in DMEM supplemented with 10% FCS. Coverslips with an even thickness

Nuclear centromere and telomere arrangements of 0.17  0.01 mm (Assistent, Germany) were used for cell attachment and growth to allow precise measurements after confocal microscopy. All cells were ¢xed and prepared for 3D-FISH according to standard protocols (Solovei et al. 2002a, 2002b). Brie£y, cells were ¢xed in 4% paraformaldehyde in 0.3  PBS (lymphocytes) or in 1  PBS (¢broblasts), permeabilized with 0.5% Triton-X100, incubated in 20% glycerol, repeatedly frozen in liquid nitrogen, and ¢nally incubated in 0.1 N HCl for 5 min. To prevent shrinkage of spherical lymphocyte nuclei, cells were brie£y (1 min) incubated in 0.3  PBS before they were ¢xed. Until hybridization, coverslips with ¢xed and pretreated cells were stored in 50% formamid/ 2  SSC at 4 C for about one week. Probes for FISH Human chromosome paint probes produced by DOP PCR from £ow-sorted chromosomes were kindly donated by M. Ferguson-Smith and J. Wienberg (University of Cambridge, UK). Chromosome paints were re-ampli¢ed by DOP PCR and depleted from repetitive sequences (Craig et al. 1997, Bolzer et al. 1999). For chromosome 18 painting, a paint probe from the respective homologue of the orangutan was kindly provided by S. Mˇller at our department. Labeling with Bio-16-dUTP, Dig-11-dUTP (Roche Molecular Biochemicals), or DNP-11-dUTP (NEN) was performed by DOP PCR. Centromere probes for chromosomes 4, 10, 11, 12, 15, 17, 18, 20, 13 þ 21, 14 þ 22 were kindly donated by M. Rocchi (University of Bari, Italy). We also employed plasmids containing an alphoid sequence speci¢c for the centromere of chromosome X (pXBR; Willard et al. 1983) and a satellite III for the paracentromeric 1q12 region (PUC 1.77; Cooke & Hindley 1979). All alphoid and satellite probes were labeled with Bio-16-dUTP or Dig-11-dUTP by nick-translation. A human pancentromeric probe (a-satellite) was generated and labeled by PCR using a27 (50 -CAT CAC AAA GAA GTT TCT GAG GCT TC) and a30 (50 -TGC ATT CAA CTC ACA GAG TTG AAC CTT CC) primers and human placenta DNA as template. Pancentromeric probes labeled with FITC-12-dUTP (Roche Molecular Biochemicals) or TAMRAdUTP (Perkin Elmer) were digested with DNase to

487 100^300 bp. A probe for mouse major satellite repeat was used as a marker of all centromere regions in mouse cells. It was generated by PCR with 50 -GCG AGA AAA CTG AAA ATC AC and 50 -TCA AGT CGT CAA GTG GAT G primers and murine genomic DNA as a template, and labeled with TAMRA-dUTP by nick-translation. Probe for telomeres was generated by PCR using (50 -TTA GGG)5 and (50 -CTT ACC)5 primers (Ijdo et al. 1991) and labeled by nick-translation with Dig-11-dUTP. 3D-FISH To preserve the 3D nuclear morphology as much as possible, air-drying of cells was carefully avoided from ¢xation through all the following steps: pretreatments, 3D-FISH, washing, and ¢nal mounting of cells in antifade (Solovei 2002b). In the case of chromosome-speci¢c paints and centromeres, labeled DNA was coprecipitated with salmon sperm DNA. Depletion of human paint probes from repetitive sequences allowed 3DFISH to be carried out in the absence of human Cot1 in the hybridization mixture (Craig et al. 1997, Bolzer et al. 1999). In some experiments, Cot1 was added as a safeguard without a notable di¡erence. Hybridization mixture in all cases consisted of 50% formamid, 10% dextran sulfate, 1  SSC. Hybridization e⁄ciency of all probes and probe combinations was ¢rst checked on metaphase spreads from normal stimulated human lymphocytes by standard 2D-FISH. For 3DFISH, a su⁄cient volume of probe was loaded onto coverslips with ¢xed and pretreated cells. A smaller coverslip was used to cover an area with cells and sealed with rubber cement. Cell and probe DNA were denatured simultaneously on a hot-block at 75 C for 2 min. Hybridization was performed for 2 or 3 days at 37 C in humid boxes. Post-hybridization washes were performed with 2  SSC at 37 C and 0.1  SSC at 60 C, respectively. Dig-11-dUTP was detected by either one layer of FITC-conjugated sheep-anti-dig antibodies (Roche Molecular Biochemicals) or two layers of mouse-anti-dig (Sigma) and Cy3-conjugated sheep-anti-mouse antibodies (Jackson ImmunoResearch Laboratories). Bio-16-dUTP was detected either by one layer of avidin-Cy3 (Jackson ImmunoResearch Laboratories) or by

488 two layers of avidin-Alexa488 (Molecular Probes) and FITC-conjugated goat-anti-avidin antibodies (Vector Laboratories). For detection of DNPdUTP, goat-anti-DNP (Sigma) and FITC-conjugated rabbit-anti-goat antibodies (Sigma) were applied. Nuclear DNA was counterstained with TO-PRO-3 (Molecular Probes) and cells were mounted in Vectashield antifade medium (Vector Laboratories). For the immunostaining of nucleoli, mouse-anti-B23 (nucleophosmin/NPM; Sigma) and Cy3-conjugated sheep-anti-mouse antibodies were applied after hybridization signal detection. Microscopy and image processing Series of light optical sections through whole nuclei were collected using a Leica TCS SP confocal system equipped with a Plan Apo 63  /1.32 NA and Plan Apo 100  /1.4 NA oil immersion objectives. For each optical section, images were collected sequentially for two or three £uorochromes. Fluorochromes were visualized using an argon laser with the excitation wavelengths of 488 nm (for Alexa 488 and FITC) and 514 nm (for Cy3), and a helium^neon laser with the excitation wavelength of 633 nm (for TO-PRO-3). Stacks of 8-bit gray-scale images were obtained with axial distances of 250^300 nm between optical sections and pixel sizes ranging from 50 to 80 nm depending on objective lens and selected zoom factor. Galleries of RGB con-focal images were assembled using NIH and Adobe Photoshop programs. Three-dimensional reconstructions of chromosome territories and their centromeres were performed by volume and surface rendering of image stacks using Amira 2.3 TGS (http://www.amiravis.com). Scoring of centromere signals RGB galleries of serial optical sections were used for visual tracing of centromere signals and scoring. Positions of speci¢c centromere signals within their chromosome territories and in the nuclei of human lymphocytes were classi¢ed as shown on Figure 1. This approach is similar to the method described by Williams et al. 2002 for classi¢cation of the gene positions in relation to the chromosome territory.

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Figure 1. Scheme for scoring of intranuclear (A) or intrachromosomal (B) positioning of the centromere signals. The position of a centromere signal in the nucleus (A) was considered as peripheral ( p, black circles) when it was touching the border of the nucleus de¢ned by counterstain or was separated from the border by a distance not exceeding centromere signal size. Other signals, including the ones adjacent to the nucleoli (n), were scored as internal (i, white circles). The arrow points at a schematic representation of a centromere cluster. In the chromosome territory (CT), represented by a shaded circle on B, all signals touching the border of a CT from inside and all ‘outside’ signals were counted as peripheral. The other signals were scored as internal.

Quantitative assessment of the 3D positioning of chromosome territories, centromeres and telomeres in lymphocyte nuclei For a quantitative 3D evaluation of CT, centromere and telomere distributions, in spherical lymphocyte nuclei, the 3D-RRD (three-dimensional Relative Radius Distribution) computer program was used (see Cremer et al. 2001 for detailed description). Brie£y, the program works as follows: (1) the gravity center of a given nucleus and its borders are determined on the basis of the nuclear counterstain signal; (2) borders of chromosome territories, centromere, or telomere signals are determined by de¢ning the threshold of the £uorescence signals in the respective color channels; (3) the nuclear radius in any direction from the nuclear center (de¢ned as the intensity gravity center of the DNA counterstain) to the segmented nuclear edge is normalized to 100% and the nuclear space is divided into 25 shells of equal thickness (each covering 4% of the total radius). In this way, the distribution of DNA (estimated as £uorescence signal intensity) of CTs, centromeres, and telomeres can be measured and expressed as a function of the relative distances of each shell from the nuclear center. Randomly distributed signals should have the same distribution as the

Nuclear centromere and telomere arrangements counterstained nuclear DNA, while deviations from the counterstain curve should indicate a nonrandom distribution. To compare relative positions of targeted structures, we calculated the average relative radius (ARR). ARR presents the mean value of the distribution of all distances from all voxels representing a signal to the gravity center of the TO-PRO-3 stained nucleus.

Results 3D analysis of centromere and CT locations in human lymphocyte nuclei Nuclear locations of centromeres detected with a pancentromeric probe Centromeres are known to cluster (Haaf & Schmid 1991, Alcobia et al. 2000). To study the degree of centromere clustering and overall distribution of the clusters in the nuclei of non-cycling human lymphocytes, hybridization with a pancentromeric probe was performed, which hybridized to a-satellite located in the central centromere domain of all human chromosomes (Mitchell et al. 1985, Choo 1997). Using this probe, we found between 7 and 18 (on average 13) signals per nucleus. No internal centromeres were found in about 60% of the nuclei (Figure 2B), while 40% contained 1^2 internal signals. The latter were always adjacent to the nucleolus. The distribution of the centromere clusters showed a maximum at the relative radius of 88^90% (green curve on Figure 2F). Locations of speci¢c centromeres with regard to their corresponding CTs To de¢ne the centromere position within its chromosome territory (CT), 3D-FISH with the chromosome paint probe and the corresponding centromere probe was performed for human chromosomes 1, 4, 11, 12, 15, 17, 18, 20, and X. Series of confocal sections were taken from the counterstained nuclei in which two homolog chromosomes and two centromere signals were clearly distinguishable after FISH. Optical sections were assembled into the RGB galleries and examined visually. Then centromere signals were classi¢ed and scored as shown on Figure 1B. In about 95% of all studied chromosomes (with the exception of the X chromosome; see below), the

489 centromere was found on the periphery of the CT (Figure 3C, D). In about 5% of the CTs studied, the positions of centromeres could be classi¢ed as internal. Only in a very few cases was the centromere signal detected at a small distance outside of the observed border of the respective CT (data not shown). Nuclear locations of speci¢c centromeres and their corresponding CTs The experiments described above were also evaluated to study the location of centromeres with respect to the nucleus. More than 80% of all centromeres were located in the immediate proximity of the nuclear border (see Figure 1A for the scoring scheme and Figure 3A, B). Lower proportions of peripheral centromeres were observed only for CTs 1 and 11 (58% and 72%, correspondingly). Most of the internally located signals were found in the immediate vicinity of a nucleolus (identi¢ed as an area stained very little, if at all, with TO-PRO-3 and surrounded by a rim of more intensely stained DNA). Surprisingly, even in the case of the NORbearing chromosome 15, the majority of the centromeres (88%) were found at the nuclear border (Figure 3A). It has been suggested that the centromeres of a signi¢cant proportion of chromosomes, including the NOR-bearing ones, are situated on the nucleoli (Carvalho et al. 2001). Correspondingly, we expected that centromeres of NOR-bearing chromosomes would be located internally together with the nucleoli. We analyzed this prediction employing 3D-FISH with three probes which hybridize to the centromere of chromosome 15 alone, to centromeres of chromosomes 13 and 21, and to centromeres of chromosomes 14 and 22. Di¡erential labeling of centromeres 13, 14, 21, and 22 was impossible because no speci¢c alphoid sequences have been identi¢ed (Choo 1997). The three centromere probes mentioned above were applied separately or as a pool, always in combination with immunostaining of the nucleolus by antibodies against nucleophosmin (B23). 3D data from approximately 150 nuclei (Table 1) revealed that G0 lymphocytes predominantly have a single nucleolus situated in the central part of the nucleus; none of the approximately 170 nucleoli abutted the nuclear border (Figure 2A). Association of

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Nuclear centromere and telomere arrangements centromere signals with the nucleolus was seen in 1.9^10.5% of the nuclei ^ never more than one signal per nucleus (Table 1). In addition to the visual examination, the 3D distributions of centromeres and their respective CTs (1, 11, 12, 15, 17, 18, 20, X) were evaluated using the 3D-RRD computer program. For each chromosome, positions of CT and centromere were quantitatively evaluated (Figure 4). For all CTs studied (red curves in Figure 4), the centromeres (green curves on Figure 4) were positioned towards the nuclear periphery, irrespective of whether the corresponding CTs were also located in the nuclear periphery (11, 12, 18, X) or more centrally (15, 17, 20). Determination of the average relative radius (ARR) of each CT showed the same trend (Figure 5): the ARR of a given centromere was always larger than the ARR of the corresponding CT. A large di¡erence between the two ARR values was noted for centrally located CTs (e.g. 17, 15). For more peripherally located CTs (e.g. 18, 11), the di¡erence was small. The ARR determined for the DNA counterstain (black line on Figure 5) varied only slightly between experiments justifying a direct comparison of the results. Location of the X-centromeres in the periphery of active X-CTs and in the interior of inactive human X-CTs In female mammalian cells, one of the two X homologs is genetically active, while the other is largely inactivated (Lyon 1961, Avner & Heard 2001). Comparison of male and female human lymphocyte nuclei showed a di¡erence in the frequency of X-centromeres located in the CT interior (Table 2). In male cell nuclei, internally located X centromeres were observed with a fre-

491 quency of 7%, similar to autosomes, while, in female cell nuclei, the incidence of internal Xcentromeres was 20% (Figure 6A, B). In human ¢broblast nuclei, this di¡erence between XX and XY genotypes was even more pronounced (Table 2). In female ¢broblast nuclei, 37.5% of all X-centromere signals were found in the X-CT interior (Figure 6C, E), while in male ¢broblast nuclei, 92% of the X-centromeres were located at the X-CT periphery (Figure 6D). In contrast to female human ¢broblast, where the active and inactive X-CT could be distinguished by morphological features, such as Barr body staining (Barr & Bertram 1949) and CT shape (Eils et al. 1996), we and others did not succeed in discriminating between the two X-CTs in female lymphocyte nuclei (M. Cremer, personal communication; Falk et al. 2002). In female ¢broblast nuclei, the centromere occupied an internal position in 54% of all evaluated Barr bodies (n ¼ 50), while only about 4% of all internal centromeres were found in active X-CTs (n ¼ 50; Table 3, Figure 6E). Centromeres of active and inactive X-CTs also di¡ered with regard to cluster formation (Figure 6F). The 3D-analysis of 21 female ¢broblast nuclei after two-color 3D FISH with probes for the X-centromere and a pancentromere probe revealed that the centromere of the inactive X was always represented by a separate signal, while the centromere of the active X clustered with centromeres of autosomes in 43% of the nuclei (Figure 6G). 3D analysis of centromere locations in murine lymphocyte nuclei To study the spatial distribution of centromeres in nuclei of murine lymphocytes, mouse major

3 Figure 2. Partial galleries of optical serial sections through a human (A^C) nucleus and a mouse (D, E) lymphocyte nucleus after 3D-FISH with centromere and telomere probes. (A) Green: FISH signals from centromeres of all human NOR-bearing chromosomes; red: nucleolus stained with antinucleophosmin antibodies; blue: nuclear DNA stained with TO-PRO-3. (B) Green: FISH signals from human pancentromere probe; red: nuclear DNA stained with TO-PRO-3. (C) Green: FISH signals from human telomeres; red: nuclear DNA. (D) Green: FISH signals from mouse pancentromeric probe; red: nuclear DNA. (E) Green: FISH signals from mouse telomeres; red: nuclear DNA; periphery. (F, G) Quantitative 3D evaluation of radial chromatin distributions in 30 human (F) and 30 murine (G) lymphocyte nuclei. Abscissa: normalized relative nuclear radius (%); ordinate: relative DNA content (%) of telomere signals (red curves), centromere signals (green curves), and TO-PRO-3 stained nuclear DNA (blue curves). n, number of evaluated nuclei in each experiment. Note a similar tendency to cluster and similar peripheral location of centromeres for both human and mouse lymphocyte nuclei. On the contrary, distribution of telomere signals differs between the two species: in human, telomere signals are distributed mainly internally, while, in mouse, telomere signals are adjacent to the chromocenters and located mainly peripherally. Bars ¼ 5 mm.

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Table 1. Spatial distribution of centromeres from NOR-bearing chromosomes

Centromeres of chromosomes

Number of observed nuclei

Average number of nucleoli per nucleus

Average number of centromere signals per nucleus

15 13 þ 21 14 þ 22

50 53 57

1.12 1.06 1.11

1.72 3.49 3.35

Number and percent of nuclei with centromeric signals abutting the nucleolus* 4 (8%) 1 (1.8%) 6 (10.5%)

*Note: The few nuclei shown in this column never showed more than one centromere signal associated with the nucleolus

satellite DNA was used. This satellite DNA is found at the pericentric heterochromatic regions of all mouse chromosomes with the exception of Y (Pardue & Gall 1970) and could be considered as a murine pancentromeric marker. Galleries of optical sections through the nuclei after 3D-FISH with murine major satellite were visually examined and, on average, 9 pericentromere heterochromatin clusters per nucleus were scored. In 33% of nuclei, only clusters adjacent to the nuclear border were found; in the other 67% of nuclei, one relatively large central cluster associated with nucleolus was present (Figure 2D). The distribution of major satellite DNA had a maximum at the relative radius of 80^85% (green curve on Figure 2G); a small plateau of the curve between 30% and 60% most probably corresponded to the nucleolus adjacent cluster. Spatial arrangements of telomeres in nuclei of human and murine lymphocytes To investigate the spatial distribution of telomeres in nuclei of both human and murine lymphocytes, a standard telomere (TTAGGG)n probe was generated by PCR. Compared with the maximum number of 92 telomere signals expected in G0

nuclei, the average number of telomere signals was only 26 in human lymphocyte nuclei, indicating a high degree of telomere clustering. It should be noted, however, that in our experiments we lacked an internal control for hybridization e⁄ciency: in contrast to the evaluation of metaphase spreads, we could not distinguish the chromosome ends. Therefore, some underestimation of the signal number cannot be excluded. Telomere signals varied in size and were located predominantly in the inner part of the nucleus (Figure 2C). On average, only 2 signals per nucleus were found at the nuclear border. Quantitative evaluation (Figure 2F) con¢rmed that telomeres tend to occupy more internal positions in human lymphocyte nuclei (red curve) than centromeres (green curve). In mouse lymphocytes, telomere clusters were often larger than in human lymphocytes (compare Figure 2E and C) and the average number of telomere signals per nucleus was 35 rather than the maximum number of 80 signals. About 57% of all telomere signals were found on the surface of chromocenters, which could be easily identi¢ed by their strong £uorescence after DNA counterstaining with TO-PRO-3 (this £uorochrome like DAPI stains preferentially the AT-rich DNA; Figure 2E). Chromocenters, which were situated

3 Figure 3. Location of centromeres in human lymphocyte nuclei (A, B) and in the respective 3D-reconstructed chromosome territories (C, D). Chromosome numbers are shown on the left. Probes represent alphoid sequences speci¢c for chromosomes 4, 11, 12, 15, 17, 18, and 20. The probe for chromosome 1 delineates the heterochromatic block, which forms the paracentromeric band 1q12. (A) For each probe two representative optical sections of the whole image stack of a typical nucleus after 3D-FISH are shown. Nuclear diameters vary depending on the axial position of an optical section. Blue: TO-PRO-3 DNA counterstain; red: painted CTS; green: signals from chromosome-speci¢c centromere probes and the 1q12 probe. (Note that false colours were chosen independent of the labeling and detection scheme.) (B) Percentage of speci¢c centromere signals with peripheral nuclear location (see the classi¢cation scheme in Figure 1A). (C) 3D reconstructions of CTs (red) shown in A with their peri- or paracentromeric heterochromatin (green). (D) Percentage of centromere signals located in the periphery of the corresponding CT. (See classi¢cation scheme in Figure 1B). Bar on A ¼ 5 mm; bars on C ¼ 1 mm.

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Figure 4. Quantitative 3D evaluation of radial chromatin distribution of painted CT and their respective centromeres in counterstained nuclei of human G0 lymphocytes. The abscissa denotes the relative radius (%) of the nuclear shells; the ordinate denotes the normalized sum of the intensities (%) of given £uorescence in a given shell. n, number of evaluated nuclei. Distributions of CT are represented by red curves; their respective centromeres by green curves; blue curves reperesent counterstained DNA. Bars indicate standard deviation of the mean for each shell.

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Figure 5. Comparison of average relative radii (ARR) of CTs and their corresponding centromere (11, 12, 15, 17, 18, 20) or paracenromere (1) subregions measured in nuclei of human G0 lymphocytes. Ordinate: ARR values for CTs (dark circles) and centromeres (white circles) are shown pairwise for each chromosome. Abscissa: pairs of ARR are arranged according to the radial distribution of a given chromosome in the nucleus starting with the most internally located CT (17: left) and ending with the most peripherally located CT (11). The black dots connected by a line show ARR-value for the TO-PRO-3 nuclear DNA counterstain in each FISH experiment.

at the nuclear periphery, carried several telomere signals mainly on the surface facing the nuclear interior. A smaller proportion of telomere signals (20%) was located directly at the nuclear border. The remaining 23% of signals were situated in the inner part of the nucleus. Localization of telomere clusters apparently correlated with their size. Telomere signals situated on peripheral chromocenters or on the nuclear border were generally large, while the internally located signals were generally small. Quantitative evaluation of £uorescence from telomere DNA showed that the distribution of the telomere signal is preferentially

peripheral and very similar to that of pericentric heterochromatin (compare red and green curves on Figure 2G).

Discussion Centromeres are located in the periphery of chromosome territories Evidence about the positions of centromeres with respect to their corresponding chromosome territories (CTs) has been sparse (Chevret et al.

Table 2. Analysis of X-centromere locations with respect of X-CTs in nuclei to male and female human G0-lymphocytes and G0-¢broblasts

Cell type and sex

Number of evaluated nuclei

Peripherally located signals (%)

Internally located signals (%)

Lymphocytes XX XY

52 30

79.8 93.3

20.2 6.7

Fibroblasts XX XY

20 25

62.5 92

37.5 8

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Figure 6. (A) Confocal section through a female human lymphocyte nucleus with two painted X-territories (red) and their respective centromeres (green). Nuclear counterstain with TO-PRO-3 is shown in blue. (B) 3D-reconstruction by surface rendering of the two X-CTs (red) shown in A demonstrates a peripheral position of the centromere (green) in the upper CT and an internal centromere position in the lower CT. (C) Mid-confocal section through a female human ¢broblast nucleus counterstained with TO-PRO-3 (white). On the left, the section is superimposed with the two 3D-reconstructed X-CTs (red) and their centromeres (green). On the right, the same confocal section shows the location of the Barr body (arrow), which corresponds to the inactive X-CT. (D) 3D-reconstructions of the two X-CTs (red) shown in C. X-CTs are shown at higher magni¢cation after surface rendering and from slightly different angles. Note the peripheral location of the split centromere (green) in the active X-CT and the internal location of the centromere in the inactive CT. (E) Mid-confocal section through a male human ¢broblast nucleus counterstained with TO-PRO-3 (white) and superimposed with its 3D-reconstructed, genetically active X-CT (red) with the X-centromere (green) at the territory periphery. (F, G) Mid-confocal section through a female human ¢broblast nucleus counterstained with TO-PRO-3 (white). The total section stack was used for the 3D-reconstruction of all centromere signals obtained by 3D-FISH with a pancentromeric probe (red) and of the two X-centromere signals (green). (F) Nuclear section with superimposed signals. One of the two X-centromere signals (green) could be assigned to the inactive X by its colocalization with the Barr body (arrow). Note: only centromeres located above the midsection are visible. (G) All centromeric signals are visible. The X-centromere signal from the active X-CT forms a cluster with the autosomal X-centromeres (dotted circle) in contrast to the X-centromere from the inactive X-CT, which typically remains as a separate entity hidden in the territory interior. Bars on A and G: 5 mm; bar on C: 10 mm applies also to E; bars on B and D: 1 mm.

2000, Nogami et al. 2000). In the present study, we employed non-stimulated human lymphocytes from peripheral blood which are in G0, and analyzed the CT-positions of the centromeres from

CTs 1, 4, 11, 12, 15, 17, 18, 20, and X. These chromosomes di¡er largely in their DNA and gene content. Our study shows that centromeres typically take a peripheral position in the respective

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497

Table 3. Location of the X-centromere in active and inactive X-CTs in female human G0-¢broblast nuclei Number of evaluated X-CTs

Peripherally located signals (%)

Internally located signals (%)

50 Inactive X-CT 50 Active X-CT

46 95.6

54 4.4

chromosome territories. An internal localization of centromeres was found in only about 2^8% of CTs. The comparison of (1) centromere locations in male and female lymphocytes and ¢broblasts, and (2) the direct comparison of centromere localization in active and inactive X-CTs in ¢broblast nuclei showed that the distribution of the centromeres in the active X-CT cannot be distinguished from autosomal CTs where *95% of all centromere signals have a peripheral positioning in the corresponding CT. In the inactive X-CT, however, the X-centromere location showed a strikingly di¡erent distribution with an internal location in about 50% of all cases. Since only very few genes are transcribed in the inactive X-CT, in contrast to the much more transcriptionally active homolog CT (Avner & Heard 2001) and the autosomes, our data suggest a correlation of centromere positions with the transcriptional activity of a given chromosome. In several studies, transcriptionally active genes were observed at the surface of CTs, while transcriptionally inactive genes were randomly distributed or rather localized in inner CT-portions (Kurz et al. 1996, Dietzel et al. 1999, Volpi et al. 2000). Other reports, however, provided evidence for the location of transcribed genes inside CTs (Abranches et al. 1998, Mahy et al. 2002a). Any interpretation of these ¢ndings should take into account the folding of CTs, which results in invaginations of the CT-surface allowing the direct contact of active genes located in the CT-interior with the interchromatin compartment (Cremer & Cremer 2001). There is strong evidence that the relocation of a number of genes in the vicinity of centromere heterochromatin contributes to their inactivation (Brown et al. 1997, 1999, Schubeler et al. 2000, Baxter et al. 2002). Our data on the common localization of centromeres in the CTperiphery in combination with the evidence for

active genes both in the CT-interior and the CTperiphery, indicate that the topology of active and inactive genes in chromosome territories is more complex than predicted in an early version of the chromosome territory^interchromatin compartment model (Zirbel et al. 1993; compare Cremer & Cremer 2001 for an up-to-date version). Matters are further complicated by interphase movements of centromeres (Ferguson & Ward 1992, Weimer et al. 1992, Vourc’h et al. 1993, Solovei et al. 2003) while we observed the highly constrained CT positions (Walter et al. 2003). The location of centromeres in the periphery of CTs may facilitate considerable movements of centromeres in the absence of CT movements. Peripheral positioning and clustering of centromeres in G0 human lymphocyte nuclei Clustering of centromeres resulting in the formation of chromocenters is typical for a wide variety of cell types (Manuelidis 1984, Haaf & Schmid 1991). The degree of clustering varies signi¢cantly between cell types and depends on the cell cycle stage (Alcobia et al. 2000, Solovei et al. 2003). Our data indicate that the transcriptional activity of a chromosome also a¡ects the ability of the centromere to cluster, though possibly indirectly. Since inactivation of the X chromosome is correlated with a shift of its centromere to the interior of the CT, centromeres of the inactive X fail to join centromere clusters. Our 3D-FISH experiments on G0 human lymphocyte nuclei demonstrate the con¢nement of centromeres from chromosomes 1, 4, 11, 12, 15, 17, 18, 20, and X to the nuclear periphery with the exception of a few cases when centromeres were found adjacent to the internally located nucleoli. Even centromeres of the NOR-bearing chromosomes (13, 14, 15, 21, 22) were strongly associated with the nuclear periphery rather than with the

498 nucleolus. These data are in general agreement with previous reports (Ferguson & Ward 1992, Weimer et al. 1992, Vourc’h et al. 1993, Skalnikova et al. 2000, Kozubek et al. 2002), although, in these studies, a considerable fraction of centromere signals was noted in the nuclear interior without an obvious association with either the nuclear envelope or the rim of the perinucleolar chromatin. In a recent study of a B-prolymphocytic leukemia derived cell line, Carvalho et al. (2001) found that centromeres of a group of largeto-medium-sized chromosomes (2, 4, 6, 7, X, 9, 10, 12) were predominantly associated with the nuclear envelope, while centromeres from a group of smaller chromosomes (15, 16, 17, 22) were often juxtaposed to nucleoli. Di¡erences between our results and these previous reports may be due to di¡erences in cell types and cell cycle stages, although di¡erences in the FISH protocols applied by us and others must also be taken into consideration. The determination of average relative radii (ARR) for each CT yielded the following sequence for the radial positioning of the studied CTs from the nuclear center towards the nuclear periphery: 17 ! 15 ! 1 ! 20 ! 12/X ! 18/11 (Figure 5). This sequence corresponds reasonably well with the sequence previously obtained by Boyle et al. (2001): 17 ! 1 ! 15 ! 20 ! X ! 12 ! 11 ! 18, although these authors used hypotonically treated, acetic-acid-¢xed and air-dried nuclei, a procedure that results in very £attened nuclei. Further, the results of our study show that the distribution of speci¢c centromeres can signi¢cantly di¡er from that of the corresponding CT. CTs 17 and 15 provide clear examples. The territory of chromosome 17, a small and gene-dense chromosome, was located in the nuclear interior (with an ARR of 58% and a modal relative radius (MRR) of 55%), while its centromere occupied a peripheral position (ARR 76%, MRR 85%). For the acrocentric chromosome 15 we also detected a distinctly peripheral position of its centromere (ARR 80%, MRR 82%), while the position of the CT was strongly shifted to the nuclear interior (ARR 65%, MRR 60%). Chromosome 15, like all other acrocentric human chromosomes, bears a nucleolus organizer region (NOR) on its short arm in the close vicinity of the 15 centromere. Since nucleoli in our study occupied a pronouncedly

C. Weierich et al. internal nuclear position, it is obvious that, in the likely case that #15-NORs contribute to the formation of active nucleoli, the short chromosome region between the centromere and the active NOR is strongly extended between the nuclear periphery (the position of the centromere) and internally located nucleolus (the position of the NOR). Our conclusion that at least a fraction of acrocentric chromosomes contributed with their NORs to the formation of internally located active nucleoli in the nuclear interior, while their centromeres were located at the nuclear periphery is re-emphasized by the ¢nding that 60% of G0 lymphocytes analyzed after 3D-FISH with a pancentromere probe revealed no internal centromere signals at all. Published data about the location of NOR-bearing chromosomes in interphase nuclei are controversial. On the one hand, it was shown that not all NORs are transcriptionally active in non-stimulated human lymphocytes, and that inactive NORs lie at a distance from the nucleoli (Wachtler et al. 1986). On the other hand, NOR-bearing human chromosomes in mouse > human cell hybrids are associated with the nucleolus, regardless of whether their ribosomal genes are transcribed or not (Sullivan et al. 2001). The proportion of centromere clusters (revealed by the pancentromeric probe) in the nuclear interior (4.5%) is lower than the proportion of internally located centromeres for some individual chromosomes (e.g. 27.8% for 11, 10^20% for 20, 17, and 15). A reason for this discrepancy is related to the clustering of the centromeres. An individually detected centromere which participated in the formation of a peripheral cluster was classi¢ed by us as an internal signal when it was adjacent to this cluster at its interior side, while the entire cluster would be classi¢ed as a peripheral one (Figure 1a, arrow; see also Alcobia et al. 2000, Figure 3). The particularly high value for chromosome 1 (41.7%) can possibly be explained by the fact that the used probe hybridized not directly to the centromere but to the paracentromeric band 1q12. While the present study was devoted to the analysis of centromere positions in non-cycling lymphocytes, several published studies targeted the distribution of centromeres at di¡erent stages of the cell cycle in human (Bartholdi 1991, Ferguson & Ward 1992, Weimer et al. 1992,

Nuclear centromere and telomere arrangements Hulspas et al. 1994) and in murine cells (Vourc’h et al. 1993). These previous studies and our own data (Solovei et al. 2003) led us to conclude that clustering of centromeres is less pronounced in nuclei of cycling than in nuclei of terminally di¡erentiated cells. Furthermore, the location of centromeres changes during the cell cycle (Ferguson & Ward 1992, Weimer et al. 1992, Vourc’h et al. 1993). In lymphocyte nuclei and several other cell types (Solovei et al. 2003), most centromeres are detected in the nuclear interior during early G1, while, in late G1, centromes move to the nuclear periphery and cluster. They remain there during early S-phase and start to de-cluster and move back to the nuclear interior in the mid^late S-phase. In late G2, most of the centromeres are again found in the nuclear interior. Telomere clusters have different locations in human and murine lymphocytes This study demonstrates that in 3D preserved human and murine lymphocytes at G0, telomeres are clustered, forming on average 26 and 35 clusters per nucleus, respectively. Accordingly, telomere clusters included on average 2.3 telomeres in mouse and 3.5 telomeres in human. Nevertheless, murine clusters were obviously larger than human ones. This ¢nding may be explained by the di¡erence in the size of telomere arrays in human and murine chromosomes (Kipling & Cooke 1990). While we used a conventional telomere FISH probe, other groups made use of a PNA telomere probe (DAKO). The PNA (peptide nucleic acid) probe was claimed to be superior with regard to signal quantitation (Zijlmans et al. 1997, Martens et al. 2000). Yet, we preferred the conventional probe, since the FISH protocol for the PNA probe provided by the manufacturer includes a strong pepsinization which we found unsuitable for 3D maintenance of the nuclear morphology. Telomere clusters were more frequent in non-cycling cells (human quiescent ¢broblasts), than in cycling and immortal HeLa cells (Nagele et al. 2001). The high degree of telomere association observed in this study in G0 lymphocytes corresponds to this ¢nding. In plants, telomeres were observed either in clusters adjacent to the nuclear periphery (Rawlins & Shaw 1990) or in clusters around the

499 nucleolus (Fransz et al. 2002). Several groups detected an accumulation of telomeres at one nuclear site and of centromeres at the opposite site, in agreement with a Rabl orientation, in both plant and animal cells (Abranches et al. 1998, Leitch 2000), though such a chromosome orientation was not necessarily found in all plants (Dong & Jiang 1998). In mouse lymphocytes Vourc’h et al. (1993) found that telomeres were distributed throughout the entire nuclear volume. Another important point is the di¡erence in the spatial distribution of telomeres which we found between human and mouse lymphocytes. Most telomeres were located in the interior of human nuclei while, in murine lymphocyte nuclei, most telomere signals, including larger clusters, were located in the periphery. For an explanation, one should consider that the mouse karyotype, in contrast to the human karyotype, consists of a set of telocentric chromosomes. Accordingly, in mouse mitotic chromosomes, half of the telomeres (the ‘proximal’ fraction) is located in the immediate vicinity of the centromeres, while the other half (the ‘distal’fraction)islocatedatthedistalendofthearm. The ‘proximal’ fraction, however, accounts only in partforalltelomereclustersfoundonchromocenters in the nuclear periphery. In telocentric mouse chromosomes (Kipling et al. 1991, Garagna et al. 2002), ‘distal’ telomeres are consistently longer than ‘proximal’ telomeres (Zijlmans et al. 1997). Therefore, the peripheral location of telomere signals of a large size suggests that ‘distal’ telomeres are also adjacent to chromocenters at the periphery of the nucleus. In conclusion, our data show both similarities and di¡erences in the spatial arrangements of centromeres and telomeres in human and murine lymphocyte nuclei. In both cases, CTs are apparently attached to the nuclear border via their pericentromeric heterochromatin, even in the case of an internal nuclear location of CTs. In human lymphocytes, p- and q-arm domains of CTs extend into the nuclear interior from peripherally located centromere clusters and terminate in the internal part of the nucleus. In mouse, our data indicate a ‘loop-like’ organization of most chromosome arm domains: they expand like human q-arms in an internal direction from peripherally located centromere/telomere clusters, but then ^ in contrast to human q-arm domains ^ return to the nucleus

500 periphery, terminating in the same or a di¡erent centromere/telomere cluster.

Acknowledgements We thank Mario Rocchi (University of Bari, Italy), Anna Jauch (University of Heidelberg, Germany), Michael Speicher and Monika Grabowski (Technical University of Munich, Germany) for providing us with human centromere probes. We are grateful to Malcolm Ferguson-Smith (University of Cambridge, UK), Johannes Wienberg and Stefan Mˇller (LMU, Munich, Germany) for their generous supply of human and monkey chromosome paints. Our thanks go also to Katrin Kˇpper and Marion Cremer from our group for their help with 3D lymphocyte ¢xation, as well as to Manuela Mohr (LMU, Munich, Germany) for providing us with mouse blood. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to T. Cremer (Cr 59/20).

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