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Blood, Vol. 95 No. 5 (March 1), 2000:
pp. 1608-1615
HEMATOPOIESIS
From the Institute of Histology and Embryology, Lisbon Medical
School, and the Nonlinear Dynamics Group, IST Department of Physics,
Lisbon, Portugal.
It is believed that the 3-dimensional organization of centromeric
heterochromatin in interphase may be of functional relevance as an
epigenetic mechanism for the regulation of gene expression. Accordingly, a likely possibility is that the centromeres that spatially associate into the heterochromatic structures (chromocenters) observed in the G1 phase of the cell cycle will differ in different cells. We sought to address this issue using, as a model, the chromocenters observed in quiescent normal human hematopoietic cells
and primary fibroblasts. To do this, we analyzed the spatial relationships among different human centromeres in 3-D preserved cells
using nonisotopic in situ hybridization and confocal microscopy. We
showed quantitatively that chromocenters in all cell types do indeed
represent nonrandom spatial associations of certain centromeres.
Furthermore, the observed patterns of centromere association indicate
that the chromocenters in these cell types are made of different
combinations of specific centromeres, that hematopoietic cells are
strikingly different from fibroblasts as to the composition of their
chromocenters and that centromeres in peripheral blood cells appear to
aggregate into distinct "myeloid" (present in monocytes and
granulocytes) and "lymphoid" (present in lymphocytes) spatial
patterns. These findings support the idea that the chromocenters formed
in the nucleus of quiescent hematopoietic cells might represent
heterochromatic nuclear compartments involved in the regulation of
cell-type-specific gene expression, further suggesting that the spatial
arrangement of centromeric heterochromatin in interphase is
ontogenically determined during hematopoietic differentiation.
(Blood. 2000;95:1608-1615)
Studies on the spatial distribution of centromeric
satellite DNA in different cell types and species have shown that
centromeres organize into 3-D patterns that appear to be cell-type
specific and evolutionarily conserved (reviewed1-9). In
mammalian cells, a tendency toward clustering of centromeres around the
nucleolus or the nuclear periphery has been consistently
observed.3 In addition, it has been shown that centromere
positioning changes during the sequential stages of the cell
cycle10 and in response to the transcriptional status of
the cell (reviewed2). In many cell types, heterochromatic
regions (mainly centromeres but also other regions of repetitive DNA)
are observed as dark-staining bodies or chromocenters that may fuse,
forming aggregate chromocenters.1,3 These structures vary
in number and size in different cell types and appear to be restricted
to the G1 phase of the cell cycle.11 All these observations
led to the proposal that centromeres may behave as structural centers
for chromatin organization in interphase, favoring the creation of
functional compartments for essential nuclear processes such as gene
expression, DNA replication, and cell division.2 Recent
observations appear to support this view because it has been shown that
repression of gene transcription can be induced by higher-order levels
of chromatin organization that spatially juxtapose the euchromatic gene
with centromeric heterochromatin.12-14 Accordingly, Brown
et al15 observed an inverse correlation of gene activity
and association with Ikaros protein, a putative transcriptional
regulator that in murine lymphoid cells surrounds constitutive
centromeric heterochromatin. These findings, together with earlier
observations of position-effect variegation phenomena in
Drosophila,16 strongly support the hypothesis that
the distribution of heterochromatin in interphase nuclei might indeed
be relevant for nuclear metabolism, namely as an epigenetic mechanism
of transcriptional regulation.14 If this is so, a likely
possibility is that centromeres belonging to specific chromosomes
should have specific patterns of spatial association in distinct
cell types.
In this study we sought to address this issue using, as a model, the
chromocenters formed during the G1 phase of the cell cycle in normal
human peripheral blood cells (lymphocytes, monocytes, and granulocytes)
and primary human fibroblasts (as representative of a nonhematopoietic
cell type). To do this, we analyzed the spatial relationships among
different human centromeres in 3-D preserved cells using nonisotopic in
situ hybridization and confocal microscopy. We showed that centromeres
in all cell types associated with a probability 2 orders of magnitude
larger than that expected for a random distribution inside the nucleus
and that the chromocenters in hematopoietic cells and fibroblasts were
made of different combinations of specific centromeres. The data thus
indicates that the spatial arrangements of centromeres in the
interphase nucleus give rise to heterochromatic compartments with
cell-type-specific composition, a fact likely to have important
implications for epigenetic regulation of tissue-specific gene expression.
Cells
Probes
In situ hybridization In situ hybridization experiments were performed as described17 with some modifications. Before hybridization, cells were repermeabilized with 0.7% Triton X-100/0.1 N HCl/phosphate-buffered saline (PBS) for 10 minutes on ice with gentle shaking, washed in PBS (3 × for 5 minutes) and 2 × SSC (1 × for 5 minutes), and denatured in 50% formamide/2 × SSC for 20 minutes at 75°C. Probes were denatured for 5 minutes at 75°C and hybridized overnight at 37°C in a moist chamber. Posthybridization washes were conducted in 50% formamide/2 × SSC at 45°C (3 × for 5 minutes). Biotin-labeled probes were detected with Texas-Red avidin (1:200; Vector Laboratories, Burlingame, CA) at 37°C for 30 minutes and washed in 0.05% Tween 20/PBS (3 × for 5 minutes). Digoxigenin-labeled probes were detected with a mouse antidigoxigenin antibody (1:100; Boehringer Mannheim) at 37°C for 30 minutes and washed as described; this was followed by incubation with a FITC-conjugated goat antimouse (1:100; Jackson Immunoresearch Laboratories, West Grove, PA). For the simultaneous visualization of hybridization sites and the nuclear envelope, the samples were incubated immediately after the detection of the hybridization signals with a rabbit anti-laminin-B antibody (kindly provided by Dr S. Georgatos, Heidelberg, Germany) diluted at 1:100 at 37°C for 30 minutes and washed as described, followed by incubation with Texas-Red conjugated goat antirabbit Ig (Jackson Immunoresearch Laboratories) at 37°C for 30 minutes.19Confocal microscopy and image analysis The analysis of hybridization signals in 3-D preserved nuclei was performed with the confocal microscope Zeiss LSM-410 (Oberkochen, Germany) as previously described.17 Twenty-five optical sections were obtained for each nucleus from hematopoietic cells (20 sections for fibroblasts) (average increment between sections in the Z axis of 0.25 µm), and the associations between different homologous or heterologous centromeres was determined in each section. To overcome problems with quantitative measures induced by distortion of confocal images, the criterion for centromere association was that of adjacency partial or total overlapping of the hybridization signals
originated by each probe. Because the nucleus of granulocytic cells
(mainly neutrophils) is polylobulated, 2 centromeres were considered
adjacent only when they were located within the same lobule (the
indentations and convolutions of the nuclei were always assessed by the
simultaneous labeling of the nuclear lamina) (see above). If
cross-hybridization between different probes was observed (this was the
case for centromeres 5 and 9, 13 and 21, and 14 and 22), single
hybridization experiments were performed and the associations between
the 4 signals originated by the individual probe were determined
(see "Results").
Statistical analysis To investigate the frequency and probability of association of different centromeres in all cell types, we used the following strategy: 100 nuclei from each cell type and for each combination of probes were analyzed. In the analysis of PBL (3100 nuclei), the combinations of probes to be used in the study were chosen so that each individual centromere was represented at least in 2 different combinations in the total data. Twelve triple combinations of probes were used (Table 1). This provided an internal control for the consistency of the data for each donor. Hybridization experiments for centromeres 2 and 3, 2 and 4, 3 and 6, 7 and 8, 7 and 9, 9 and 11, 15 and 16, 15 and 17, and 16 and 17 were further performed in cells from 4 different donors. Probabilities have been calculated within a universe of events in the range 100 to 301. When each of the 2 centromeres from homologous chromosomes in an individual cell were associated with a different centromere, these associations were considered as 2 independent outcomes. To further assess the consistency of the data, a few pairs of centromeres whose frequencies of association (either frequent or rare) could be predicted on the basis of the results obtained in 1 donor were analyzed in the same or different donors. For example, if triple combinations of probes specific for centromeres A, B, C and B, C, D showed that A is associated with B and B is associated with D, then a double hybridization experiment was performed with probes for A and D to see whether the 2 centromeres were also associated, as should have been expected from the previous results. The same reasoning was applied for some low-association expected frequencies (see "Results"). In quiescent monocytes, granulocytes, and fibroblasts, the analysis was specifically focused on selected pairs of centromeres that had been found to be frequently associated (or less frequently associated) in lymphocytes (double-hybridization experiments) (Table 1). To investigate whether the observed results would fit a putative model for a random association of centromeres within the nucleus, the areas of the median optical section of each nucleus and of the hybridization signal were measured. The nuclei of lymphocytes have a mean radius in the order of r0 = 3 µm3, and the fluorescent signals are approximately circular with a radius of r1 = 0.2 µm3. Therefore, if we consider that the PBL nuclei are spherical and have a radius r0 and if the position of centromeres in interphase is random, then the probability of touching or overlapping of 2 signals of the same size is given by P = (2 r1/r0)3 = 0.0024.
Chromocenters in quiescent peripheral blood cells and primary fibroblasts To determine whether centromeres aggregate to chromocenters in quiescent human cells, a pan-centromeric probe was first hybridized to 3-D preserved unstimulated peripheral blood cells and primary fibroblasts in G1. The results in the former showed that instead of the 46 single dot signals that should be expected for normal diploid human cells in G1, the hybridization signals merged into irregular masses of various sizes and shapes, whose number varied from 6 to 18 (mean, 11) per nucleus in lymphocytes, 13 to 28 (mean, 21) in granulocytes, and 6 to 20 (mean, 11) in monocytes and were predominantly associated to the nuclear envelope or the nucleolus (see Figure 1 and examples in Figure 2A). This differed from what was observed in PBL stimulated with phytohemagglutinin, in which the number of signals was consistently higher (more than 35) and more single-dotted in most of the cells (not shown). As previously reported,11 aggregation of centromeres was also observed in quiescent fibroblasts. However, the hybridization patterns differed from those in unstimulated lymphocytes by the higher number of hybridization signals (mean, 25 per nucleus) (Figure 2B). In summary, the data show that centromeres do aggregate to heterochromatic structures in unstimulated peripheral blood lymphocytes, monocytes, and granulocytes, which, in the case of lymphocytes, disperse after stimulation with a mitogen and are larger than those observed in quiescent fibroblasts.
Patterns of centromere associations in chromocenters of peripheral blood cells and fibroblasts We next sought to investigate whether these structures represented random associations of different centromeres or the preferential clustering of specific centromeres in each cell type. To do this, probes specific for each of the human centromeres were first hybridized to quiescent lymphocytes. As shown in Table 1, different centromeres have different frequencies of association. The highest are between centromeres of the acrocentric chromosomes (pairs 13-21 and 14-22, associated in more than 60% of the cells), which were almost always in proximity to the nucleolus (as visualized by the superimposition of fluorescent and phase-contrast images), followed by centromeres 15 and 16 (46% ± 0.02%), the latter also in proximity to the nucleolus; 10 and 11 (42%); 18 and 20 (41%); 7 and 9 (43% ± 0.03%); 8 and 9 (38%); 15 and 17 (38% ± 0.03%); 11 and 12 (37%); and 17 and 18 (34%) (see example in Figure 3). Associations were consistently observed between heterologous centromeres because those from homologous chromosomes were rarely associated (less than 2% of the cells in each experiment for each pair of homologs). To verify whether the observed associations varied among different donors, hybridization experiments with probes for a few pairs of centromeres that had been found to have higher (centromeres 7 and 8, 7 and 9, 15 and 16, 15 and 17, 16 and 17) or lower (centromeres 2 and 3, 2 and 4, 3 and 6) frequencies of association in 1 donor were subsequently performed in samples derived from 4 other donors. No significant differences were observed among different donors for the more-associated or the less-associated pairs of centromeres (all P > 0.05) (Table 1; cases with mean values ± SD). To further verify the consistency of the data, pairs of centromeres whose patterns of association could be predicted from the analysis using triple combinations of probes were subsequently analyzed in cells from different donors in double-hybridization experiments. More specifically, triple hybridization for centromeres 1-2-3 showed that associations between centromeres 1 and 2 occurred in 18% of the cells, whereas the combination of probes for centromeres 2-3-4 showed a mean association between centromeres 2 and 4 of 20%. Double hybridization with probes for centromeres 1 and 4 was then performed and showed an association of 19% for this pair of centromeres. Similarly, the triple combinations of probes for centromeres 2-3-4 and 3-4-6 showed a frequency of association of 16% and 15% for centromeres 2 and 3 and 3 and 6, respectively. Double hybridization with probes for centromeres 2 and 6 in another donor was 18%. The triple combinations for centromeres 7-8-9 and 9-10-11 showed frequencies of associations of 43% for centromeres 7-9 and of 35% for centromeres 9-11. Centromeres 7 and 11 were found to be associated in 30% of the cells from a different donor (Table 1). Probabilities of association between pairs of centromeres were found to be the same for these double- and triple-hybridization experiments. They had an error of 1% except for centromere pair 7 and 8, in which the error of association was 5%. Taken together, the data show that the association patterns between specific centromeres are maintained in cells derived from different donors.
In this study we investigated the spatial association of centromeres
into chromocenters of quiescent human peripheral blood cells and
fibroblasts. The data show that these interphase heterochromatic structures correspond to nonrandom spatial arrangements of specific centromeres in all cell types, that the chromocenters observed in these
cells are made of different combinations of centromeres, and that, as
to the emerging patterns of centromere associations, fibroblasts are
strikingly different from hematopoietic cells, and, within the latter,
cells of myeloid origin (monocytes and granulocytes) are similar to
each other and different from lymphoid cells.
The authors thank M. Carmo-Fonseca for critical review of the manuscript.
Submitted August 23, 1999; accepted November 1, 1999.
Supported by a grant from Program PRAXIS XXI. IA was supported by a
PRAXIS XXI fellowship.
Reprints: Leonor Parreira, Instituto de Histologia e
Embriologia, Faculdade de Medicina de Lisboa, Avenida Prof. Egas Moniz
1699, Lisboa Codex, Portugal; e-mail: hleonor{at}correio.fm.ul.pt.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
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Top
Abstract
Introduction
-DNA sequences. Two main
findings emerged from the study: first, the observed frequencies of
association showed that the centromeres close to the nucleolus mostly
belonged, as expected, to chromosomes containing nucleolar organizers
(NOR). However, the nucleolus-related clusters of centromeres were not
exclusive of NOR-containing chromosomes because the centromeres of
chromosomes 16 (which does not contain a NOR) were frequently associated with those of chromosome 15, near the nucleolus, a fact that
might be related to the presence of large constitutive pericentromeric
heterochromatic blocks in chromosome 16.
