Chromosome Research 11: 485^502, 2003.
# 2003 Kluwer Academic Publishers. Printed in the Netherlands
485
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,
C. Weierich et al.
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.
C. Weierich et al.
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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C. Weierich et al.
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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C. Weierich et al.
Nuclear centromere and telomere arrangements
493
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.
494
C. Weierich et al.
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.
Nuclear centromere and telomere arrangements
495
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
496
C. Weierich et al.
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
Nuclear centromere and telomere arrangements
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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