Download Imprinted SNRPN within chromosome territories

2157
Journal of Cell Science 113, 2157-2165 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS1007
Relative locations of the centromere and imprinted SNRPN gene within
chromosome 15 territories during the cell cycle in HL60 cells
Masahiro Nogami1,2, Atsushi Kohda1, Hiroshi Taguchi1, Mitsuyoshi Nakao3, Toshimichi Ikemura2 and
Katsuzumi Okumura1,*
1Laboratory of Molecular and Cellular Biology, Faculty of Bioresources, Mie University, 1515 Kamihama, Tsu, Mie 514-8507,
2Department of Evolutionary Genetics, National Institute of Genetics, and The Graduate University for Advanced Studies,
Japan
Mishima, Shizuoka 411-8540, Japan
3Department of Tumor Genetics and Biology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan
*Author for correspondence (e-mail: [email protected])
Accepted 24 March; published on WWW 25 May 2000
SUMMARY
Investigations of imprinted regions provide clues that
increase our understanding of the regulation of gene
functions at higher order chromosomal domains. Here, the
relative positions of the chromosome 15 centromere and the
imprinted SNRPN gene in interphase nuclei of human
myeloid leukemia HL60 cells were compared, because the
homologous association of this imprinted chromosomal
domain was previously observed in lymphocytes and
lymphoblasts. Four targets including the chromosome 15
territory, its centromere, the SNRPN gene on this
chromosome, and the nucleus, were visualized
simultaneously in three-dimensionally preserved nuclei
using multicolor fluorescence in situ hybridization, and the
spatial distributions of these probes were analyzed with
a cooled CCD camera deconvolution system. We found that
preferential association of SNRPN interhomologues did
not occur during the cell cycle in HL60 cells, although this
gene exhibited asynchronous replication and monoallelic
expression in this cells. SNRPN was found to localize at the
periphery of the chromosome territories, and it
preferentially faced the nuclear membrane, unlike the
adjacent centromeric repeat. The SNRPN gene and the
centromere were located close to each other late in S phase,
reflecting that these DNA segments may be compacted into
the same intranuclear subcompartments with the progress
of S phase and in course of preparation for the following
G2 phase. Our results suggest that, although an imprinted
gene has features similar to those observed with
intranuclear localization of other gene coding sequences,
the characteristic of mutual recognition of imprinted
regions is determined by certain cellular regulation, and it
is not necessary for the allele-specific features of an
imprinted gene.
INTRODUCTION
interphase nuclei by fluorescence in situ hybridization (FISH;
Cremer et al., 1988; Lichter et al., 1988; Pinkel et al., 1988). In
mammalian cells, interphase chromosomes are generally
considered to be less condensed than their mitotic counterparts,
but individual chromosomes occupy discrete compartments
called chromosome territories that do not overlap each other
(Cremer et al., 1993; Strouboulis and Wolffe, 1996; Bridger
and Bickmore, 1998). Sub-chromosomal regions, such as
chromosome bands, are also compartmentalized during
interphase (Visser et al., 1998; Jackson and Pombo, 1998; Zink
et al., 1999), suggesting that the structures of condensed
metaphase chromosomes are reflective of their organization in
interphase nuclear space. Coding sequences are known to be
located at the surfaces of their respective chromosome territories
independent of their transcriptional status (Kurz et al., 1996).
The relationships between gene activities and three-dimensional
(3-D) morphologies of chromosome territories were examined
with a set of active and inactive human X interphase
chromosomes. Active X territories were found to have flatter
shapes and larger, more irregular surfaces in comparison to the
Genomic imprinting marks the parental origin of chromosomes
and results in allele-specific changes in expression, chromatin
structure, and replication (Surani, 1994; Efstratiadis, 1994;
Razin and Cedar, 1994; Barlow, 1995). Many imprinted genes
are clustered, and these regions are thought to be regulated by
higher order chromosomal domains (Nakao and Sasaki, 1996).
Therefore, investigation of the behavior of these domains
provides insight into the mechanisms that regulate genomic
imprinting. One approach toward such purposes is to visualize
target genes and domains in the nucleus three-dimensionally
and examine their nuclear dynamics under various conditions.
Recent advances in digital imaging technology, confocal
microscopy, and deconvolution systems have made it possible to
analyze the nuclear organization of specific genomic sequences
(Agard et al., 1989; Ferguson and Ward, 1992). Information
about the nuclear architecture is accumulating gradually.
Chromosome painting probes can be used to visualize directly
the territorial organization of each specific chromosome in
Key words: Multicolor FISH, Chromosome territory, SNRPN,
Genomic imprinting, HL60 cell
2158 M. Nogami and others
smoother surfaces and rounder shapes of inactive X territories
(Eils et al., 1996). Comparison of human chromosomes 18 and
19, which are of similar sizes showed that genome organization
affects the localization and morphology of chromosomes in the
nucleus (Croft et al., 1999). Some investigators have examined
the complexity and variability of the shapes of chromosome
territories. They analyzed the spatial relationships between
chromosome territories and splicing machinery or the
localization of DNA replication sites in the territories. Splicing
machinery components are concentrated in between
chromosome territories, and DNA replication foci are located in
the interiors of territories (Zirbel et al., 1993; Clemson et al.,
1996; Zink et al., 1998; Verschure et al., 1999). Since the
patterns of DNA replication foci change in a cell cycledependent manner (Nakayasu and Berezney, 1989; Fox et al.,
1991; O’Keefe et al., 1992) and expression of individual DNA
segment is cell-type specific, it is also important to examine
organization of genomic DNA in the nucleus in relation with the
cell cycle and cell type.
Observations about the dynamics of nuclear genome
organization in somatic cells and the relationships to biological
phenomena have been reported. The positions of centromeres
and telomeres during the cell cycle are dynamic (Ferguson
and Ward, 1992; Vourc’h et al., 1993). Human acrocentric
chromosomes that contain rDNA sequences are well known
to be components of nucleoli. Alterations of nuclear
architecture might be associated with the changes observed
in tumorigenesis (Linares-Cruz et al., 1998). Although the
number of examples studied is small, these observations
support the idea that transcription and genomic sequences have
implications for nuclear architecture, intranuclear structure and
behavior of chromosomes. From this point of view, it is
interesting to examine how intranuclear genome organization
changes in biological phenomena, such as genomic imprinting.
In the present study, we focused on an imprinted gene,
SNRPN (small nuclear ribonucleoprotein polypeptide N),
because homologous association of the different alleles of this
imprinted gene domain occurs late in S phase during the cell
cycle (LaSalle and Lalande, 1996). This gene is located on
human chromosome 15q11-q13, and one candidate gene for
the Prader-Willi syndrome/Angelman syndrome (PWS/AS;
Horsthemke, 1997). LaSalle and Lalande found the allelic
segments in this domain were closer than the chromosome
15 centromere homologues. They suggested that normal
imprinting involves preferential association of the
interhomologues of this gene region. We compared the relative
positions of the chromosome 15 centromere and the SNRPN
gene in interphase nuclei of human myeloid leukemia HL60
cells, because this cell line is well cultured and expected to
maintain imprinting aspects of the SNRPN gene. First, we
examined whether the association of homologous alleles of
SNRPN was observed or not during the cell cycle in HL60
cells, after we confirmed asynchronous replication and
monoallelic expression of this gene in HL60 cells.
Furthermore, the relative positions of these loci during the cell
cycle were analyzed with respect to the territories occupied by
all of chromosome 15 to examine whether an imprinted gene
has features similar to those observed with intranuclear
localization of gene coding sequences. So far as we know, this
is the first report that examines the relationship between an
imprinted gene and a chromosome territory.
MATERIALS AND METHODS
Chemicals and DNA probes
RPMI1640, fetal bovine serum (FBS), and human Cot-1 DNA (GibcoBRL); bromodeoxyuridine (BrdU), 4′,6-diamidino-2-phenylindole
(DAPI), diazacyclooctan (DABCO), and propidium iodide (PI;
Sigma); anti-BrdU monoclonal antibody and anti-mouse antibodies
conjugated with either FITC or rhodamine (MBL); and anti-biotin
conjugated with Cy-5 (Jackson Lab.) were purchased from the
indicated sources. Reagents used for probe labeling and signal
detection for FISH were purchased from Boehringer Mannheim.
Other chemicals were from Nacalai Tesque unless otherwise stated.
Cosmid 93 (c93) spanning the SNRPN region and pPR541 in MHC
locus were kindly provided by A. L. Beaudet (Baylor College of
Medicine; Sutcliffe et al., 1994) and H. Inoko (Tokai Univ. School of
Medicine), respectively. A centromere probe, pCM15, was kindly
provided by A. Baldini (Baylor College of Medicine). Cy-3conjugated chromosome 15 painting probe was purchased from
Cambio (Cambridge, UK).
Cell culture and preparation of specimens
Human myeloid leukemia HL60 cells were cultured in RPMI1640
containing 10% FBS at 37°C in an atmosphere of 5% CO2. The HL60
cells were fractionated by centrifugal elutriation. Cultured cells (3×108
cells) were loaded onto a Beckman elutriator rotor at 2,000 rpm at 4°C
with flow of 0.3% gelatin in PBS (phosphate-buffered saline). The
flow rate was increased from 11 ml/minute to 26 ml/minute at every
2.5 ml/minute increment. For each flow rate, the initial 100 ml were
collected, and aliquots of each fraction were analyzed by flow
cytometry (FACSCalibur; Becton Dickinson). Fractionated cells were
immediately fixed by the method of Kurz et al. (1996) with slight
modification. Briefly, cells were suspended in PBS and fixed with 4%
paraformaldehyde in PBS for 10 minutes at room temperature. After
treating with 1% paraformaldehyde in 0.1 M HCl for 10 minutes, the
fixed cells were permeabilized with PBS containing 0.5% Triton
X-100, 0.5% saponin, and 1% paraformaldehyde for 10 minutes.
Before each treatment, cells were harvested at 200 g at 4°C for 5
minutes, and the supernatant was removed. The permeabilized cells
were equilibrated with 20% glycerol in PBS for 20 minutes and stored
in liquid nitrogen until use.
FISH
FISH was performed essentially as described previously (Lichter et
al., 1988). Cells frozen in liquid nitrogen were washed twice with
PBS. The cells were mounted onto a poly-D-lysine-coated coverslip
and placed on it for 15 minutes for the fixation. Cells were equilibrated
with 2× SSC for 10 minutes and then further equilibrated with
denaturing solution (50% formamide in 2× SSC) for 15 minutes. Cells
were then equilibrated with the hybridization solution for at least 20
minutes prior to denaturation. The nick-translated cosmid
(biotinylated) and centromere (directly fluorescein labeled) probes
were mixed with 3 µg of Cot-1 DNA and denatured at 80°C for 10
minutes in the hybridization solution (50% formamide (v/v), 10%
dextran sulfate in 2× SSC) and preannealed at 37°C for 30 minutes.
The chromosome 15 painting probe was treated at 42°C for 20
minutes and mixed with the preannealed probes just before
hybridization. The cells on the coverslip were denatured for 10
minutes at 80°C, and then the above hybridization probe mixture was
added. Hybridization was carried out overnight at 37°C in a moist
chamber. Each coverslip was washed three times with 50% formamide
in 2× SSC for 5 minutes at 42°C and then three times with 2× SSC
at 60°C. The coverslips were then incubated for 30 minutes in a
blocking solution (3% Block Ace (Yukijirushi), 0.1% Tween-20 in 2×
SSC) at 37°C and detected with Cy-5-conjugated avidin (in 1% Block
Ace, 2× SSC, 0.1% Tween-20) for 30 minutes at 37°C. Then,
coverslips were washed three times with 4× SSC, 0.1% Tween-20 for
Imprinted SNRPN within chromosome territories 2159
5 minutes at 42°C and counterstained using 0.2 µg/ml DAPI in 2×
SSC for 5 minutes. Finally, the coverslips were mounted on whole
slide glasses with an antifade solution (Vectashield; Vector
Laboratories) and sealed with nail polish. Using this protocol, the
SNRPN gene, chromosome 15 centromere, chromosome 15 territory,
and nucleus were detected by Cy-5, fluorescein, Cy-3, and DAPI,
respectively.
FISH-based replication assay
Replication timing of cosmid clones was determined by FISH (Selig
et al., 1992) with slight modification. A biotinylated probe was
hybridized to methanol-acetic acid-fixed nuclei and detected with
fluorescein-avidin. S phase nuclei were simultaneously visualized by
incorporated BrdU following detection steps with anti-BrdU and
rhodamine-conjugated antibodies. Slides were counterstained with
DAPI and examined with 63× oil objective on a Zeiss Axioskop
fluorescence microscope fitted with a Zeiss filter set for DAPI, FITC,
and rhodamine. Signals were observed in more than 95% of interphase
nuclei. Only slides with strong FISH signals and low background were
scored. At least 200 S phase nuclei were scored, and their signal
patterns classified as singlet-singlet (SS), singlet-doublet (SD), or
doublet-doublet (DD). Only clear round signals were scored as
singlets. Extremely strong, large signals, and signals with rod-like or
oval-like shapes were classified as doublets. The maximal difference
between the two examiners was 2.0% for SD%, and their data were
averaged.
RNA-FISH
The intron probes for the RNA-FISH analysis were generated by PCR.
PCR was carried out using c93 as template and the following primer
sets: (5′-AGGAAAAGGAACAGGGTAAGGGAAA-3′, 5′-GCAGACGCAGCAGAGGTGACAG-3′, 5′-ACGCAACACAGACCCCCAGG-3′, 5′-GGAAGGGCGGTGGTGACTGG-3′) corresponding to
approximately 3 kb sequences on the upstream of exon alpha of the
SNRPN gene. PCR products were labeled by nick translation using
digoxigenin-11-dUTP. The RNA-FISH protocol was using previously
described (Dirks and Raap, 1995), with modifications. HL60 cells
were fixed with 4% paraformaldehyde and 5% acetic acid for 20
minutes at room temperature. The cells were washed three times for
5 minutes with PBS and stored in 70% ethanol at −20°C. Cells were
fixed onto a poly-D-lysine-coated coverslip and treated by 0.01%
pepsin in 0.01% HCl for 5 minutes at 37°C, followed by a short wash
in water and fixed in 1% paraformaldehyde for 5 minutes at room
temperature, then washed with PBS, dehydrated in a 70, 90 and 100%
ethanol series and air-dried. The hybridization mixture containing
labeled intron probe and salmon sperm DNA (10 µg) in hybridization
solution was mounted on the coverslip. Hybridization was carried out
at 37°C overnight in a moist chamber. The coverslips were washed
four times with 50% formamide in 2× SSC for 3 minutes at 37°C, and
once in 2× SSC for 3 minutes at room temperature. The hybridized
probe was detected with rhodamine-conjugated anti-digoxigenin Fab
fragment and followed by blocking step. Finally, the coverslip was
washed three times with 0.1% Tween-20 in 4× SSC for 2 minutes at
37°C, then counterstained with DAPI.
Optical sectioning and 3-D-image analysis
Series of light optical sections of nuclei were recorded with a cooled
CCD camera (PentaMax 1317K-1, Princeton Instruments) mounted
on a Zeiss Axioplan 2 MOT microscope with a Plan-Apochromat
63×/1.40 oil objective controlled by IPLab Spectrum software (Signal
Analytics) on a Macintosh 9600/200MP computer. The Ludle filter
wheel systems were equipped with both excitation and emission filters
that were also controlled by IPLab. For sectioning, the microscope
stage was lowered at 0.1 µm (input) intervals, and at each focal plane,
the four fluorescent colors were imaged through respective filter
combinations. The sectioned images of each color were deconvoluted
separately using HazeBuster 2.0 software (VayTek) to remove haze,
and the 3-D images were reconstructed using extension utilities of
IPLab Spectrum and VoxBlast 2.01(VayTek). These 3-D reconstructed
images were used to confirm the positioning of the probes and the
territories. Signal classification in the chromosome territory was based
on the merged sectioned images at each focal plane. Because the
detectable sensitivity between chromosome painting probes and
probes for specific sequences is different, FISH signals are often
observed outside the chromosome territories (territory cores).
Therefore, as shown in Fig. 2b, the signal was classified as located at
the periphery of a territory (territory core) if the center of the signal
was outside or at the boundary of the visible territory; on the other
hand, the signal was considered within a territory if the center of the
FISH signal was inside the visible border of the territory (territory
core). In this method, classification of FISH signals at the boundary
of the territory may be thought to be ambiguous. However, differences
between two examiners was not significant. The following results are
based on the analyses of 74 to 94 nuclei in each fraction during the
cell cycle.
Statistical analysis
To test whether the different distributions of organization patterns are
significant or not, P (probability) was calculated. All the significant
results obtained in the present study were within 99.9% confidence
levels (P≤0.001) except for one result (99% level, P =0.01).
RESULTS
Replication timing and expression of SNRPN in
HL60 cells
Before studying the intranuclear positioning of the SNRPN
gene, we examined whether this gene has general properties of
genomic imprinting, i.e. asynchronous replication and
monoallelic expression. First, the replication timing profile of
the SNRPN gene was determined by FISH-based assay. As
shown in Fig. 1A, the SNRPN gene exhibited more than 25%
of asynchronous pattern (SD), although the SD% of a standard
clone pPR541 was less than 10%. This confirms that the
SNRPN replicates asynchronously in HL60 cells. Since the
cells in S phase were selected by simultaneous detection of
BrdU with different color combination with the probe, SS%
and (SS + SD)% reflect the approximate time of replication of
the first allele and the second allele in S phase, respectively.
More than 60% for SS signal indicates that both alleles of the
SNRPN gene replicate during the later half of S phase in HL60
cells.
Next, RNA-FISH using intron probes of the SNRPN gene
was carried out to visualize the primary transcript in the
nucleus. As shown Fig. 1B, most nuclei (79.6%) exhibited only
one spot, suggesting that the imprinted expression of this gene
is maintained in HL60 cells, although we could not determine
which parental allele of this gene was actively transcribed.
Cell fractionation and 3-D FISH imaging
To study the spatial arrangement of specific genomic sequences
in the nucleus, SNRPN gene, chromosome 15 centromere,
whole chromosome 15, and the nucleus were studied by
multicolor FISH. A cosmid containing the SNRPN gene was
labeled with biotin-dUTP and detected by anti-biotin
conjugated Cy-5. The centromere probe was labeled with
fluorescein-dUTP. To detect chromosome 15 in its entirely, we
used a commercially-available Cy-3-conjugated painting
probe. The q arm of chromosome 15 was stained
2160 M. Nogami and others
Fig. 1. Analysis of replication timing and expression of the SNRPN
gene. (A) FISH-based replication timing assay was performed to
show the replication asynchrony of SNRPN. FISH signals of the
cosmid c93 (including SNRPN sequence) or a standard clone pPR541
in S phase nuclei were classified as SS, SD, or DD. The higher SD%
of SNRPN indicates that this gene replicates asynchronously between
the homologous alleles. (B) RNA-FISH to detect the primary
transcripts of SNRPN gene were carried out using the intron probes
generated by PCR. The representative image was captured by a
deconvolution system. To demonstrate all the intranuclear RNA
signals, three focal planes with 0.3 µm (input) intervals of RNA
signals were imaged and merged with one of the same focal planes of
the DAPI image. About 80% of nuclei were detected only one signal,
indicating that the monoallelic expression of SNRPN is maintained in
HL60 cells.
homogeneously by this probe, indicating that it was suitable
for the delineation of chromosome territories in the nucleus
(data not shown).
The above three probes were hybridized to
paraformaldehyde-fixed nuclei and detected by the respective
color, within the nuclei which were stained with DAPI. An
optical section was taken every 0.1 µm (input) for each nucleus
(total 60-80 sections). Typical pseudocolored and merged
images of every third focal planes with FISH signals are shown
in Fig. 2a. To determine the intraterritorial locations of each
fluorescence signal, only the necessary images were merged,
for example, SNRPN and the territory, or the centromere and
territory. In Fig. 2b-left, one focal plane merged with two
probes and territory is shown as an example. The locations of
the signal centers of each probe on the same focal plane are
examined (Fig. 2b-right). The center of the centromere probe
(marked green +) is detected within the visible border of the
chromosome territories, whereas that of the SNRPN probe
(marked blue +) is located at the periphery or outside of the
territory. Because of the differences in sensitivity between the
painting probe and the specific sequences, probes are often
observed outside the territory (territory core). Therefore, the
signal whose center was observed inside the territory core was
considered as internal localization; the signal whose center
was observed outside or boundary the territory core was
considered as peripheral localization. All the sectioned images
for each nucleus were surveyed to determine the classification
of the FISH signals. Three-dimensional reconstruction of
FISH images was performed with VoxBlast software. A
representative 3-D image was shown in Fig. 2c. The 3-D
images were used to confirm the positioning between probes
and territories. Nucleoli are visible in the nuclei as dark regions
that are not stained with DAPI. It is interesting to delineate
nucleoli, because human chromosome 15 is acrocentric, and
the rDNA genes located on the p arm of this chromosome may
be associated with nucleoli. In fact, many FISH signals were
observed near and around the nucleoli (data not shown).
To investigate cell-cycle dependent localizations of genomic
sequences in the nucleus, cultured HL60 cells were
fractionated by centrifugal elutriation. This method was used
instead of cell synchronization to avoid the effects of drugs
necessary to control the cell cycle. Four fractions were pooled,
and aliquots from each fraction were analyzed for the cell cycle
profile by flow cytometry. As shown in Fig. 3, fractions A, B,
C, and D were rich in G1, S, late S, and G2/M phase cells,
respectively. Because M phase cells in the G2/M rich fraction
were excluded from these analyses, the fractions were
designated G1, S, late S, and G2. After the elutriation, cells
were fixed immediately in paraformaldehyde as described
previously (Kurz et al., 1996). We used a protocol that did not
include alcoholic and protease treatments to better preserve the
intranuclear organization of the genomic sequences.
Preferential association of SNRPN homologues
does not occur in HL60 cells
LaSalle and Lalande (1996) reported homologous association
of the oppositely imprinted human chromosome 15q11-q13
region in late S phase. They observed a marked increase in the
number of nuclei with closely-associated 15q11-q13 region
(within 2 µm) in late S phase, even compared with the
chromosome 15 centromere. They also showed that the
imprinted status is important for this association. Since SNRPN
in this locus has been confirmed to show monoallelic
expression and asynchronous replication in HL60 cells
(Fig. 1A and B), we expected that association of SNRPN
homologues would be observed during late S phase in most
nuclei. Therefore, we compared the interhomologue distance
of SNRPN with that of the centromere in HL60 cells for the
SNRPN and centromeric probes, we scored the number of
nuclei in which the distance between the one pair of the
homologues was closer than the distance between the other pair
in the same nucleus (Fig. 4). The specific association of
SNRPN homologues in late S phase was not significant
statistically (P>0.3). The results showed that there were no
significant differences in relative distances during the cell cycle
for either probe. Although HL60 cells maintain imprinting of
SNRPN, we could not find any specific association of this
Imprinted SNRPN within chromosome territories 2161
locus. In contrast, interhomologues of both SNRPN and the
centromere were observed in the nuclear peripheries and apart
from each other in most cases (data not shown).
Distribution of chromosome 15 centromeres or
SNRPN genes within the chromosome territories
Although the number of examples are not many, gene-coding
sequences were shown to be located at the surface of the
chromosome territories independent of their transcriptional
activities, and non-coding sequences were found randomly
distributed or localized in the interior of the chromosome
territories (Kurz et al., 1996). Here, the distributions of the
chromosome 15 centromere and SNRPN gene within the
chromosome territories were examined for each fraction of the
cell cycle, to know whether these DNA segments have such
features or not. It is also interesting to compare the intranuclear
positioning of physically adjacent DNA segments with
different properties, because the human SNRPN gene is located
on chromosome 15q11-q13 close to the centromere, and both
of these target sequences are spaced closely as detected by
FISH. The images in Fig. 5 are examples of deconvoluted,
merged images on the same focal plane. Only patterns for the
SNRPN signal is demonstrated for easier view. The relative
position of SNRPN or the centromere within the chromosome
territory was examined on the sectioned images, and the
number of territories with the patterns in the upper three
images in the figure (a-c) were scored. The image (a) shows
the FISH signal of SNRPN (or the centromere) probe located
on the periphery of the chromosome territory and facing the
nuclear membrane. In the lower part of this figure, the
frequency of this pattern to the total number of cells scored is
calculated and shown as a red bar for each cell fraction. The
image (b) shows a FISH signal located on the periphery of the
territory and facing the interior of the nucleus. The percentage
Fig. 2. Three-dimensional analysis of the centromere and SNRPN within chromosome 15 territories. (a) Examples of optical section images by
a deconvolution system. Sixty to eighty sections obtained every 0.1 µm (input) were collected for each nucleus. The four fluorescent signals
were imaged in each focal plane using a filter wheel system. Each set of collected images was deconvoluted, and the deconvoluted images on
the same focal plane were merged and pseudocolored. Chromosome 15 painting probe (Cy-3; red), c93 SNRPN probe (Cy-5; blue), pCM15
centromere probe (fluorescein; yellow/green), and DAPI-stained DNA (gray). Fifteen images with 0.3 µm (input) intervals of the representative
nucleus are shown in the figure. (b-left) Example of a chromosome territory (red) after merging of the centromere (yellow/green) and SNRPN
(blue) images acquired from the same focal plane. (b-right) Same image as in b-left is represented in gray scale for the territory. Centers of the
centromere and SNRPN signals are marked as green and blue (+), respectively, to determine their intraterritorial localizations. In this case, the
centromere and SNRPN was considered as interior and periphery of the territory, respectively, because the center of the centromere is inside the
territory, but that of SNRPN is at the boundary or exterior the territory. (c) Representative 3-D image generated by a deconvolution system. The
same pseudocolors as the above are used. The left image was rotated counter-clockwise from the z-axis (c-right).
2162 M. Nogami and others
Cell number
A
B
C
D
700
700
700
700
280
280
280
280
0
0
0
200
400
600
0
0
200
400
600
0
0
200
400
600
0
200
400
600
Relative DNA content
A
G1
S
G2
83
11
6
49
22
B
41
5
C
4
D
0
29
54
68
28
20
40
60
80
1 0 0 ( %)
Fig. 3. Cell-cycle profiles of HL60 cells fractionated by centrifugal elutriation. Human myeloid leukemia HL60 cells were cultured in
RPMI1640 containing 10% FBS. 1-5×108 cells were elutriated with 0.3% gelatin in PBS. Aliquots of each fraction were analyzed by flow
cytometry (FACSCalibur). (A-D) Cell-cycle profile of each fraction. A-D are G1, S, late S, and G2 rich fraction, respectively. The percentage of
each cell cycle stage, G1, S, and G2/M, is also shown.
of cells with this pattern is indicated as an orange bar in the
figure. The image (c) is a pattern that the FISH signal is
detected in the territorial volume. This percentage is shown as
a blue bar in the figure. As clearly shown, the centromere probe
preferentially localizes in the territorial volume during the cell
cycle (blue, average, 79%). The SNRPN gene localized in the
more peripheral part of the chromosome territory than the
centromere independent of the cell cycle stage (P<0.0001). The
average was 56% for SNRPN but 21% for the centromere.
These results are consistent with the findings of Kurz et al.
(1996). Allelic differences in the positions within the territories
were not evident (data not shown). Furthermore, the SNRPN
gene preferentially faces the nuclear membrane particularly in
the late S and G2 phases. For SNRPN, 58% and 71% of the
FISH signals located on the periphery of the chromosome
territories face the nuclear membrane in the late S and G2
phases, respectively, while these values are 32% and 37% for
the centromere (P>0.6 for G1 and S; P=0.01 for late S; P=0.001
for G2). Relative positions of the SNRPN
and centromere FISH signals in the nucleus
were not significant during the cell cycle
(data not shown).
SNRPN
centromere
G1
( n= 8 7 )
22
S
( n= 7 4 )
21
0
34
55
25
42
24
( n= 9 4 )
42
45
20
late S ( n= 7 9 )
G2
36
20
40
34
60
80
1 0 0 ( %)
Fig. 4. Comparison of the relative interhomologue distances between the SNRPN and the
centromere. The patterns shown in the top of the figure were scored and each percentage
is indicated. The black bar is the ratio of the pattern that the distance of the SNRPN
homologues is shorter than that of the centromere ones. The white bar is the opposite
pattern. The hatched bar is the ambiguous pattern. (n) indicates the number of nuclei
analyzed. Statistic analysis indicates that preferential association of SNRPN homologues
was not significant in the late S phase in HL60 cells, as compared with that of the
centromere (P>0.3).
Cell cycle-dependent association of
SNRPN and chromosome 15
centromere in HL60 cells
The distance between the SNRPN and
centromere signals was compared across the
cell cycle. As shown in Fig. 6, four FISH
signal patterns for SNRPN and the
centromere were observed: (a) both probes
colocalized, (b) both probes overlapped, (c)
both probes were separated but were within
one signal distance, or (d) both probes were
separated by more than one signal distance.
Both the FISH signals were close to each
other during late S and G2 phases, as
compared with G1 and S phases (P≤0.001,
Fig. 5; purple and orange bars). This result
suggests that these sequences are associated
in late S phase. This may be more
understandable if it is taken into
Imprinted SNRPN within chromosome territories 2163
consideration that the late S fraction was not well-fractionated
and that the G2 fraction contains many cells in late S phase (see
Fig. 3). Because the map positions of SNRPN and the
centromere are close on chromosome 15, this genomic region
may be more extended in early S and more condensed in late
S phase. It is possible that these DNA segments are compacted
into the same intranuclear subdomains in the course of
preparation for the normal G2 phase.
DISCUSSION
We analyzed the cell-cycle dependent spatial arrangement of
the imprinted SNRPN gene on human chromosome 15 in
human myeloid leukemia HL60 cells, because this gene is on
a well-characterized chromosomal domain, the PWS/AS
critical region, and a relationship between the genomic
function and the intranuclear behavior has been suggested
(LaSalle and Lalande, 1996). We asked, first of all, whether the
imprinted status of SNRPN is necessary and sufficient for the
homologous association of this gene locus. As shown in Fig.
1, HL60 cells were confirmed to maintain its monoallelic
expression and asynchronous replication, which are general
properties of imprinted genes. We expected that this gene pairs
between homologues in a specific stage of the cell cycle as
previously observed. However, in the present study, the
association of homologous alleles of SNRPN was not observed
during the cell cycle in HL60 cells (Fig. 4). The results of this
study contradict a report of homologous association of this
imprinted chromosomal domain in lymphocytes and
lymphoblasts (LaSalle and Lalande, 1996). Our results suggest
that the characteristic mutual recognition of imprinted regions
is determined by specific cellular regulation, and is not
necessary for the allele-specific features of an imprinted gene.
Another group also found preferential pairing of the imprinted
region on distal mouse chromosome 7 in fibroblast cells
(Riesselmann and Haaf, 1999), although their observation was
not so significant. Long-time cultured cells like HL60 might
lose this sort of specific features of imprinted domains.
Next, we collected general observations on the relative
positions of gene-coding and repetitive sequences within the
chromosome territories or the nuclei using the chromosome
15 centromere and SNRPN as examples. We delineated four
targets stained with different colors simultaneously with a
deconvolution system. In contrast with the centromere, we
found that the SNRPN gene is localized in the periphery of
the chromosome territories and preferentially faces the
nuclear membrane in late S and G2 phases (Fig. 5). The
peripheral localization of SNRPN in the chromosome
territories was consistent with the findings of Kurz et al.
(1996) that genes are located at the surface of their respective
chromosome territories independent of their transcriptional
status. Although this gene shows imprinted expression, we
could not find allelic differences in position within the
territories in individual nuclei (data not shown). Localization
of a gene and its primary transcript near the nuclear
membrane may be responsible for prompt transport of the
processed RNA through the nuclear pores. DNA replication
may be another explanation for the peripheral localization
because both alleles of SNRPN replicate asynchronously but
late in S phase (Gunaratne et al., 1995, Fig. 1A), and DNA
replication usually occurs at the periphery of the nucleus in
the later half of S phase.
The boundaries of territories and nuclei are an important
point. Kurz et al. (1996) characterized active and inactive genes
by considering a visible border of the territory exterior region.
Eils et al. (1996) analyzed the shape and surface structures of
the active and inactive X chromosome territories using 3-D
Vorunoi tessellation procedure. In HL60 cells, the shape of the
nucleus varies. Therefore, to examine the homologous
association, it was considered more accurate to compare the
relative positions of two signals than to determine the absolute
distance of FISH signals from the center or the edge of the
nucleus. The shapes of territories are also complex and
variable, and the surfaces of territories are not smooth. Even if
a gene was observed within a territory, some factors could be
accessible to it through a hollow on the territories (Clemson et
al., 1996). The difference in sensitivities between the cosmid
and the painting probes should also be considered. In this point
of view, it may be reasonable to regard the painted regions as
territorial cores, because FISH signals of SNRPN are often
observed outside the painted territories. In fact, we determined
intraterritorial localization of the probes based on this territory
core (Fig. 2b).
We observed that SNRPN and the centromere are located
close to each other particularly late in S phase (Fig. 6). Since
SNRPN maps to chromosome 15q11-q13, it is close to the
centromere. Therefore, it is not surprising that their FISH
signals are adjacent to each other in interphase nuclei; in fact,
approximately 90% of the signals were detected within one
signal distance (see the green bars in Fig. 6). However, as
shown in Fig. 6, 49% and 44% of both probes were associated
with or overlapped each other during late S and G2 phases,
respectively. This is very high because late S fraction contains
many cells in G2 stage and vice versa (see Fig. 3), indicating
that this genomic region is more extended in early S phase and
more condensed in late S phase. Although it has been
suggested that the organization of chromatin segments within
a chromosome territory is random (van den Engh et al., 1992;
Yokota et al., 1995), these two loci may be compacted into the
same intranuclear sub-compartments with the progress of S
phase. The assembly of these loci into a compartment after
DNA replication may be in preparation for the following
normal G2 and division stage. In recent model, DNA
polymerases are immobilized by attachment to larger
structures, and concentrated in discrete ‘replication factories’,
where they reel in their templates and extrude newly made
nucleic acids (Cook, 1999). Generally, a large number of small
foci are observed early in S phase, while a relatively small
number of assembled foci are observed in the middle and late
S phases (Nakayasu and Berezney, 1989; Fox et al., 1991;
O’Keefe et al., 1992). Since both alleles of SNRPN and the
centromere replicate in the later half of S phase, it might be
possible that both regions assemble into the same replication
domains before or after DNA replication. In late S phase,
replicating regions and flanking-replicated regions might be
collected into larger replication factories. To clarify the
meaning of this compartmentalization, it is necessary to
examine the positioning of these probes in relations to various
intranuclear structures.
As demonstrated in Fig. 1, SNRPN imprinted expression and
asynchronous replication are maintained in HL60 cells.
2164 M. Nogami and others
Fig. 5. Distribution of SNRPN or the centromere within the chromosome territories in HL60 cells. The upper three images, which are
deconvoluted focal planes, show the typical FISH signal pattern of the SNRPN probe in the chromosome territory as examples; (a-c) probe at
territory periphery and facing nuclear membrane, probe at territory periphery and facing away from membrane, probe inside of territory. The
percentage of each localization pattern observed in each cell cycle fraction is shown in the figure as a colored bar corresponding to the frame
color. The SNRPN gene is localized in the more peripheral part of the chromosome territory than the centromere independent of the cell cycle
stage (P<0.0001) and this gene preferentially faces the nuclear membrane particularly in the late S and G2 phases, as compared with the
centromere (P>0.6 for G1 and S; P=0.01 for late S; P=0.001 for G2). n, number of samples analyzed.
Although SNRPN and the centromere tend to colocalize or
overlap in late S phase, a number of cells showed differences
of FISH signal patterns in the homologues during the cell
cycle. In 30 to 40% of the nuclei, SNRPN and the centromere
on one chromosome were colocalized, while they were
separated on the other chromosome (data not shown).
Unfortunately, polymorphic markers to identify the alleles did
not work in this cells (Kitsberg et al., 1993; Ritchie et al.,
1998). Although the parental origins of the alleles were not
determined in this study, these results may reflect allelic
differences in the chromatin structure of this imprinted region.
As mentioned before, allelic differences in intraterritorial
locations of SNRPN was not found. So, this allelic difference
may be correlated with DNA replication timing.
Imprinted regions are thought
to be regulated by higher-order
chromosomal domains (Nakao
and Sasaki, 1996). Therefore, it
is interesting how intranuclear
genome organization is related to
this biological phenomenon. The
present study is a first step to
clarifying this important biological
problem as well as to understanding
that the intranuclear behaviors
of the genomic sequences have
biological significance.
Fig. 6. Cell-cycle dependent changes in the distance between SNRPN and the centromere on the
same chromosome. Patterns indicated at the top of the figure were scored, and the percentage of
each is indicated; (a-d) both probes colocalize, both probes overlapped, both probes separated but
within one signal distance, both probes well separated. The percentage of each pattern observed in
each cell cycle fraction is shown in the figure as a colored bar corresponding to the frame color. In
each image, SNRPN and the centromere are blue and green, respectively. Both probes were close
to each other during late S and G2 phases, as compared with G1 and S phases (P≤0.001). ‘n’ in
parentheses indicates the number of signals analyzed.
We thank M. Lalande (University of
Connecticut School of Medicine) for
a polymorphic marker of PWS/AS
region. We also thank H. Inoko (Tokai
Univ. School of medicine), and A.
Baldini and A. L. Beaudet (Baylor
College of Medicine) for kindly
providing a cosmid pPR541, human
chromosome 15 centromere and the
SNRPN cosmid, respectively. We are
grateful to Shin Ogata for the
centrifugal elutriation and FCM
analyses. A part of this work is
performed by a NIG Cooperative
Imprinted SNRPN within chromosome territories 2165
Research Program (‘98-29). Support was provided in part by a Grantin-Aid for Creative Basic Research; a Grant-in-Aid for Scientific
Research on Priority Areas; a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science, Sports, and Culture of Japan,
and a Grant-in-Aid for Special Coordination Funds for Promoting
Science and Technology from the Science and Technology Agency of
Japan to K.O.
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