COMMENTARY
Three-dimensional arrangements of chromatin and chromosomes: old
concepts and new techniques
R. APPELS
CSIRO Division of Plant Industry, Black Mountain, GPO Box 1600, Canberra, Australia
Introduction
The studies of early cytologists, well before the turn of
this century, indicated that the positions of mitotic
chromosomes in a particular metaphase cell correlated
with their respective positions in preceding cell divisions.
Observations of this type led to the concept that metaphase chromosomes were not simply aggregations of
dispersed chromatin. Furthermore, when it became clear
that chromosomes were the carriers of the genetic material many ideas developed relating the arrangement of
chromosomes to gene expression in nuclei. A major
problem in analysing interphase nuclei was the difficulty
in visualizing chromosomes at this stage in the cell cycle.
Recent advances in combining the techniques of molecular biology with modifications to the optical microscope
and computer image enhancement, analysis and interpretation have revolutionized the capability of studying the very old problem of whether order exists within
the eukaryote nucleus.
In a recent issue of J. Cell Science, D. J. Rawlins and
P. J. Shaw (1988) analysed the positions of chromosomes
relative to each other in anaphase root-tip cells of Crepis
capillaris; this plant has only three chromosome pairs.
The authors fixed root-tip cells in 4 % formaldehyde and
studied 47 mitotic cells in anaphase (stained with the
fluorescent dye DAPI) using the new technology of
optical sectioning and computer image analysis mentioned above. In this material, hpwever, they were unable
to find any evidence of particular arrangements of the
chromosomes that were favoured over others. In this
issue, J. L. Oud, G. J. Brakenhoff, H. T. M. van der
Voort, E. A. van Spronsen and N. Nanninga, present a study of the same biological material fixed in ethanol: acetic acid (3: 1, v/v), using similar technology. The
larger number (75) of mitotic cells in anaphase studied by
Oud et al. revealed a significant surplus of an arrangement of anaphase chromosomes that placed the two
nucleolar organizer chromosomes next to each other.
Overall, the two studies both showed that relatively few
specific arrangements of chromosomes were favored in
anaphase mitotic divisions. The tendency of the nucleolar
organizer (Nor locus) chromosomes to be positioned close
Journal of Cell Science 92, 325-328 (1989)
Printed in Great Britain © The Company of Biologists Limited 1989
to each other is of special interest, however, because the
nucleolus is such a major structure in the nucleus.
Nucleolar Dominance
The phenomenon of nucleolar dominance is observed
when cells have more than one nucleolus organizer but
only one of these is transcriptionally active. A. J. Hilliker
& R. Appels (unpublished data) have argued that nucleolar dominance is a reflection of the juxtapositioning of
chromosomes carrying the Nor loci. The authors suggested that the usual 'state' of the rDNA was inactive due to
the binding of a repressor(s) and that this resulted in its
cytological appearance as heterochromatin. The competition between Nor loci for a limited pool of activator
molecules was then postulated to occur when the loci
were replicated and temporarily free of repressor molecules. In this way only a small portion of the cell cycle
would be available for determining the subsequent state
of activity of a given Nor locus. At all other stages of the
cell cycle the RNA polymerase I and/or transcription
factors would not be able to interact with the inactive Nor
loci and only the activated loci would be transcribed.
Hilliker & Appels (unpublished) suggested that the
activator molecule may be either a diffusible molecule,
produced at a specific time in the cell cycle and capable of
cooperative binding to rDNA, or a part of the nuclear
matrix/membrane that attains a suitable conformation
for binding rDNA at a certain stage in the cell cycle. The
model for the structural arrangement of the nucleolus
shown in Fig. 1 is well suited to the idea that juxtapositioned Nor loci would compete with each other for a point
of attachment to the nucleoskeleton (protein matrix) or
nuclear membrane and thus for eventual transcriptional
activity.
The rDNA locus is a very large one, especially in
plants where the tandemly arranged units can amount to
18000 kb of DNA, and the attachment to a fixed component of the nucleus is potentially a reasonable mechanism for explaining the nucleolar dominance phenomenon.
325
quences in nuclei could be measured the model would be
directly testable.
The Measurements of the Positions of Specific
DNA Sequences in Unsquashed Nuclei
Preparations
Transient attachment of
spacer DNA to protein matrix
Protein matrix
(includes RNA polymerase I)
I
Fibrillar centre
I
Fibrillar component
Fig. 1. Model for an active nucleolus. The model shown is
based on those of Sommerville (1984), Bureau et al. (1986)
and Deltour & Mosen (1987), and suggests that a key point of
interaction between the protein matrix of the fibrillar centre
and rDNA is via the repetitive units of the spacer region.
The granular component that surrounds the fibrillar
component is not shown. The suppressed nucleolus has its
rDNA units compacted into heterochromatin and therefore
unable to take up the extended conformation required for
activity.
The corollary of this model is that two or more rDNA loci
must be physically close to each other in order to compete
for a binding site that is not diffusible. The time at which
these regions are replicated at a particular site on the
nuclear membrane (or nucleoskeleton, see Cook, 1988)
could conceivably identify a period during which they are
near enough to each other to compete for a nearby
binding site. Such a critical three-dimensional arrangement of chromosomes would probably exist for only a
short period of the cell cycle and the Nor loci not bound to
the activator during this period would become inactive
shortly thereafter.
The model accounts for the observation that plasmids
carrying segments of rDNA are freely transcribed after
introduction into cells (Vance et al. 1985; Grummt &
Skinner, 1985; Grimaldi & DiNocera, 1986; Clos et al.
1986; Kuhn & Grummt, 1987). According to the model
the introduced plasmids are not within the region of the
chromosome that takes up an inactive conformation
(involving non-diffusible repressors resulting in a heterochromatic appearance). The plasmids are therefore not
repressed
and thus free to utilize the RNA polymerase
rep
I/transcription
factors available. The simpler model for
l/t>
nucleolar dominance based on competition for a free, but
limited, pool of RNA polymerase I (or other transcription factors) cannot readily account for the transcription
of rDNA plasmids in a cellular environment where Nor
competition is occurring.
The nucleolar dominance model highlights two aspects
of chromosome arrangements in nuclei, which will be
discussed further in the following paragraphs. First, the
model presented introduces the possibility that critical
chromosome juxtapositions may be transitory; and
second, that if the distribution of specific rDNA se326
R. Appels
The techniques used by Rawlins & Shaw (1988) and Oud
et al. (1989) for assaying the positions of anaphase
chromosomes have also been applied to the analysis of
nuclei after the hybridization (in situ) of cloned DNA
probes. The details of the techniques and their application to the study of Drosophila salivary chromosome
bands have been described by Rykowski et al. (1988). An
important advance in the area of charting the positions of
specific chromosomes in interphase nuclei has been the
isolation of chromosome-specific repetitive probes (for
studying rye chromosomes in wheat backgrounds, see
Appels et al. 1978, 1986; for studying human chromosomes, see references quoted by Trask et al. 1988;
Cremer et al. 1988). Pinkel et al. (1989) have developed
the use of DNA probes to a high level of chromosomal
selectivity by combining mixed probes from chromosome-specific DNA libraries with the addition of an
excess of total human DNA to 'compete out' unwanted
signals from repetitive sequences. This permits the
'staining' of the entire chromosome. The use of fluorescent probes has, furthermore, permitted the quantification of the amount of probe bound (Pinkel et al. 1986;
Hiraoka et al. 1987) and the computer image analyses of
the type discussed by Rykowski et al. (1988), Trask et al.
(1988) and Manuelidis & Borden (1988).
The application of the technologies described above to
the positioning of interphase chromosomes relative to
each other, or to an easily recognizable cytological
structure, has been published in the last 12 months
(Trask et al. 1988; Manuelidis & Borden, 1988). Hopman
et al. (1986) demonstrated the feasibility of simultaneously detecting two different DNA sequences in situ
that hybridize to probes labelled with either mercury or
biotin. Trask et al. (1988) reported the use of two
different fluorescent probes to locate chromosome 17 and
the Y chromosome simultaneously in a three-dimensional
reconstruction of a single nucleus; these authors did not
describe any statistical study of the relationship between
the positions of the chromosomes in the nuclei of the cell
types used. Manuelidis & Borden (1988) examined differentiated cell types (neurones) and found specific associations between chromosomes 1 and 9 and the nucleolus;
in approximately 30% of nuclei both chromosome 1
signals were associated with the nucleolus, while for
chromosome 9 this situation occurred in 60 % of the
nuclei. In the remaining nuclei a single signal from the
respective chromosomes was associated with the nucleolus. In only 5 % of nuclei was there no association
between these chromosomes and the nucleolus. In control experiments with dispersed sequences it was demonstrated that many other chromosomal regions are distributed in areas away from the nucleolus. It is thus apparent
from the study by Manuelidis & Borden (1988) (see also
Hadlaczky et al. 1986) that in a particular state of
differentiation of a cell some chromosomes are positioned
in recognizable regions of the nucleus.
Metaphase Chromosome Positions
Prior to the recent advances in optical sectioning major
approaches to studying chromosome arrangements involved the analysis of serially sectioned tissues by electron microscopy and metaphase chromosome spreads.
The work showed that polyploidy or interspecies hybrids
in plants resulted in separation of the chromosomes in
nuclei according to their origin (Avivi et al. 1982; Finch
etal. 1981; Bennett, 1984; Schwarzacher-Robinson et al.
1987). In the F l hybrid between Secale and Hordeum
species, for example, the difference in size between the
two sets of chromosomes was sufficient to allow Bennett
and his colleagues to conclude that they remain separate
many divisions after the initial formation of the embryo.
In animals, similar conclusions have been reached by
workers studying metaphase chromosome spreads after
C-banding (Coates & Wilson, 1985; Zelesco & Graves,
1988). An important development from these studies was
the recognition of the need for a statistical analysis of
available data to validate significant relationships between
chromosomes (Callow, 1985). Computer analysis of data
relating to chromosome or chromatin arrangements in
nuclei is a rapidly expanding area of research and the
optical sectioning techniques are providing the appropriate numbers of observations for statistical studies.
indicated that the only chromosomal regions that were
cross-linked originated from the same arm of a chromosome. Essentially no cross-links were found between
different chromosome arms, leading to the conclusion
that chromosomes were in clearly defined domains even
in interphase when they are in an apparently diffuse state.
Conclusions
The combination of in situ hybridization with the new
technology of optical sectioning and image analysis is
providing unique insights into the arrangement of
chromatin in nuclei. The problem of artefacts generated
by the experimental procedures is always present but
changes in the technique, such as the enzymic production
of single-stranded DNA rather than harsh denaturation
steps (van Dekken et al. 1988), will help to reduce this
problem. Classical problems, such as nucleolar dominance, will hopefully be re-examined by using specific
DNA sequence probes to shed new light on very old
observations and ideas.
References
APPELS, R., DRISCOLL, C. J. & PEACOCK, W. J. (1978).
Heterochromatin and highly repeated DNA sequences in rye
(Secale cereale). Chromosoma 70, 67-89.
APPELS, R., MCINTYRE, C. L., CLARKE, B. C. & MAY, C. E. (1986).
Alien chromatin in wheat: ribosomal DNA spacer probes for
detecting specific nucleolus organizer region loci introduced into
wheat. Can. J. Genet. Cytol. 28, 665-672.
AVIVI, L., FELDMAN, M. & BROWN, M. (1982). An ordered
Chromosome Domains
The three-dimensional reconstruction experiments using
in situ hybridization of chromosome-specific probes have
shown that at interphase chromosomes remain in relatively compact regions within the nucleus. Homologous
chromosomes appear to have no tendency to be located
near each other in anaphase of mitosis (Rawlins & Shaw,
1988; Oud et al. 1989) or in interphase nuclei (Trask et
al. 1988; Manuelidis & Borden, 1988). In studies on the
organization of chromatin in eukaryote nuclei problems
related to fixation artefacts need to be assessed (Cook,
1988), and it is thus important that different experimental approaches yield similar conclusions. The conclusions
referred to above regarding chromosome domains were,
for example, also obtained from cytogenetic studies by
Hilliker (1986). Hilliker and his colleagues have studied
the relative chromosome positions in Drosophila melanogaster cells that are destined to differentiate into salivary
gland cells. The assay measuring the relative positions of
chromosomes included the irradiation of larvae at a
growth stage when progenitor salivary gland cells have
undergone their last cell division. The irradiation treatment caused cross-linking between chromosome regions
that are juxtaposed. The actual positions of the crosslinks could be accurately measured cytologically by
studying the polytene chromosomes of the fully differentiated salivary gland cells. These cytogenetic studies
arrangement of chromosomes in the somatic nucleus of common
wheat. I. Spatial relationships between chromosomes of the same
genome. Chromosoma 86, 1-26.
BENNETT, M. D. (1984). Nuclear architecture and its manipulation.
Stadler Symp. 16, 469-502.
BUREAU, J., HUBERT, J. & BOUTEILLE, M. (1986). Attachment of
DNA to nucleolar and nuclear skeletal structures as visualized by
Kleinschmidt molecular spreading. Biol. Cell 56, 7-16.
CALLOW, R. S. (1985). Comments on Bennett's model of somatic
chromosome disposition. Heredity 54, 171-177.
CLOS, J., NORMANN, A., OHRLEIN, A. & GRUMMT, 1. (1986). The
core promoter of mouse rDNA consists of two functionally distinct
domains. Nucl. Acids Res. 14, 7581-7595.
COATES, D. J. & WILSON, D. (1984). The spatial distribution of
chromosomes in metaphase neuroblast cells from subspecific Fl
hybrids of the grasshopper Caledia captiva. Chromosoma 90,
338-348.
COOK, P. R. (1988). The nucleoskeleton: artefact, passive framework
or active site. J. Cell Sci. 90, 1-6.
CREMER, T., TESIN, D., HOPMAN, A. H. N. & MANUELIDIS, L.
(1988). Rapid interphase and metaphase assessment of specific
chromosomal changes in neuroectodermal tumor cells by in situ
hybridization with chemically modified DNA probes. Expl Cell
Res. 176, 199-220.
DELTOUR, R. & MOSEN, H. (1987). Proposals for the macromolecular
organization of the higher plant nucleonema. Biol. Cell 60, 75-85.
FINCH, R. A., SMITH, J. B. & BENNETT, M. D. (1981). Hordeum and
Secale genomes lie apart in a hybrid. J. Cell Sci. 52, 391-403.
GRIMALDI, G. & Di NOCERA, P. P. (1986). Transient expression of
Drosophila melanogaster rDNA promoter into cultured Drosophila
cells. Nucl. Acids Res. 14, 6417-6432.
GRUMMT, I. & SKINNER, J. A. (1985). Efficient transcription of a
protein-coding gene from the RNA polymerase 1 promoter in
transfected cells. Pwc. natn. Acad. Sci'. U.S.A. 82, 722-726.
HADLACZKY, G., WENT, M. & RINGERTZ, N. R. (1986). Direct
Arrangements of chromatin and chromosomes
327
evidence for the non-random localization of mammalian
chromosomes in the interphase nucleus. Expl Cell Res. 167, 1-15.
HlLLlKER, A. J. (1986). Assaying chromosome arrangement in
embryonic interphase nuclei of Drosophila melanogaster by
radiation-induced interchanges. Genet. Res. 47, 13-18.
RAWLINS, D. J. & SHAW, P. J. (1988). Three-dimensional
HlRAOKA, Y., SEDAT, J. W. & AGARD, D. A. (1987). The use of a
RYKOWSKI, M. C , PARMELEE, S. J., AGARD, D. A. & SEDAT, J. W.
charge-coupled device for quantitative optical microscopy of
biological structure. Science 238, 36-41.
HOPMAN, A. H. N., WIEGANT, J., RAAP, A. K., LANDEGENT, J. E.,
analysis using quantitative, high-sensitivity, fluorescent
hybridization. Proc. natn. Acad. Sci. U.SA. 83, 2934-2938.
organization of chromosomes of Crepis capillaris by optical
tomography. J. Cell Sci. 91, 401-414.
(1988). Precise determination of the molecular limits of a polytene
chromosome band: regulatory sequences for the Notch gene are in
the interband. Cell 54, 461-472.
VAN DER PLOEG, M. & VAN DUIJN, P. (1986). Bi-color detection of
two target DNA's by non-radioactive in situ hybridization.
Histochemistry 85, 1-4.
KUHN, A. & GRUMMT, I. (1987). A novel promoter in the mouse
rDNA spacer is active in vivo and in vitiv. EMBO J. 6,
3487-3492.
SCHWARZACHER-ROBINSON, T . , FlNCH, R. A . , SMITH, J. B. &
MANUELIDIS, L. & BORDEN, J. (1988). Reproducible
TRASK, B., VAN DEN ENGH, G., PINKEL, D., MULLIKIN, J.,
WALDMAN, F., VAN DEKKEN, H. & GRAY, J. (1988). Fluorescence
compartmentalization of individual chromosome domains in human
CNS cells revealed by in situ hybridization and three-dimensional
reconstruction. Chromosoma 96, 397-410.
OUD, J. L., BRAKENHOFF, G. J., VAN DER VOORT, H. T. M., VAN
SPRONSEN, E. A. & NANNINGA, N. (1989). Three-dimensional
arrangement of anaphase chromosomes of Crepis capillaris root tip
cells as studied by confocalscanning Laser microscopy. J. Cell Sci.
92, 329-339.
PINKEL, D., LANDEGENT, J., COLLINS, C , FUSCOE, J., SEGRAVES,
R., LUCAS, J. & GRAY, J. (1989). Fluorescence in situ
hybridization with human chromosome specific libraries: Detection
of trisomy 21 and translocations of chromosome 4. Ptvc. natn.
Acad. Sci. U.S.A. 85, 9138-9142.
PINKEL, D., STRAUME, T. & GRAY, J. W. (1986). Cytogenetic
328
R. Appels
BENNETT, M. D. (1987). Genotypic control of centromere positions
of parental genomes in Hordeum X Secale hvbrid metaphases. J.
Cell Sci. 87, 291-304.
SOMMERVILLE, J. (1985). Organizing the nucleolus. Nature, Lond.
318, 410-411.
in situ hybridisation to interphase cell nuclei in suspension allows
flow cytometric analysis of chromosome content and microscopic
analysis of nuclear organization. Hum. Genet. 78, 251-259.
VANCE, V. B., THOMPSON, E. A. & BOWMAN, L. H. (1985).
Transfection of mouse ribosomal DNA into rat cells: faithful
transcription and processing. Nucl. Acids Res. 13, 7499-7513.
VAN DEKKEN, H., PINKEL, D., MULLIKIN, J. & GRAY, J. W. (1988).
Enzymatic production of single-stranded DNA as a target for
fluorescent in situ hybridization. Chromosoma 97, 1-5.
ZELESCO, P. A. & GRAVES, J. A. M. (1988). Chromosome
segregation from cell hybrids. IV. Movement and position of
segregant set chromosomes in early phase interspecific cell hybrids.
J. Cell Sci. 89, 49-56.