Chapter 2

Interphase Chromosome Behavior in Normal

and Diseased Cells
Gemma Bourne, Catherine Moir, Ural Bikkul, Mai Hassan Ahmed,
Ian R. Kill, Christopher H. Eskiw, Sabrina Tosi, and Joanna M. Bridger

Abstract Interphase chromosomes are nonrandomly positioned in the nuclei of

normal cells. They occupy specific locations with respect to a radial distribution
from the nuclear edge to the nuclear interior. Furthermore, there is some evidence
that interphase chromosomes reproducibly have the same neighbors that can be
involved in creating translocations which lead to cancer. Not only are chromosomes
nonrandomly positioned but they are anchored to certain regions of the cell nucleus
by cellular structures such as the nuclear lamina and the nucleolus. Global screening
of the genome has identified both lamina-associated domains and nucleolarassociated domains. Increasingly, researchers are finding that interphase chromosomes are mislocalized in disease situations. The consequences of chromosome
mislocalization are not yet that clear, but gene expression can be affected with interphase chromosomes being located in another compartment of the nucleus, changing
G. Bourne I.R. Kill
Laboratory of Cellular Gerontology, Centre for Cell and Chromosome Biology (CCCB),
Brunel University, Kingston Lane, West London UB8 3PH, UK
C. Moir C.H. Eskiw
Laboratory of Molecular Gerontology, CCCB, Brunel University, Kingston Lane,
West London UB8 3PH, UK
U. Bikkul
Laboratory of Cellular Gerontology, CCCB, Brunel University, Kingston Lane,
West London UB8 3PH, UK
Laboratory for Nuclear and Genomic Health, CCCB, Brunel University, Kingston Lane,
West London UB8 3PH, UK
M.H. Ahmed J.M. Bridger (*)
Laboratory for Nuclear and Genomic Health, CCCB, Brunel University, Kingston Lane,
West London UB8 3PH, UK
e-mail: [Link]@[Link]
S. Tosi
Leukaemia and Chromosome Research Laboratory, CCCB, Biosciences, Brunel University,
Kingston Lane, Uxbridge UB8 3PH, UK
Y.B. Yurov et al. (eds.), Human Interphase Chromosomes: Biomedical Aspects,
DOI 10.1007/978-1-4614-6558-4_2, Springer Science+Business Media, LLC 2013

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G. Bourne et al.

their interactions with nuclear structures. This chapter outlines how chromosomes
behave in interphase nuclei and with what they interact. We discuss many examples
of when chromosomes, and the genes housed upon them, change their location and
behavior in disease situations such as cancer and the premature aging syndrome
called HutchinsonGilford progeria syndrome. We also describe new findings
whereby genes in the host are relocated and expressed after a parasitic infection.

Introduction
The eukaryotic cell nucleus is a highly complex organelle that contains the cell
genome in the form of interphase chromosome territories. Interphase chromosomes
are described thus because as they decondense, after cell division within the new
daughter nuclei, they remain in and interact with a particular area of the nucleus, a
so-called territory of the nucleus. Although there is some intermingling and coming together of genes from different chromosomes, most of the body of one chromosome is maintained together, and thus to all intents and purposes chromosomes
in interphase are separate entities. The interphase chromosomes are nonrandomly
positioned in nuclei, revealing that there must be a high level of genome reorganization post mitosis to obtain individual chromosome territories in the right compartments of the nucleus (Fig. 2.1).
The structures within nuclei are intimately involved in organizing and positioning
interphase chromosomes to allow the coordination of a wide range of functions centred
around the genome, such as gene expression and silencing, splicing and processing,
and DNA replication and DNA repair. These nuclear structures are all linked and are
part of a functionally responsive cellular network (Starr 2009). Such architecture comprises the nuclear envelope with all its components: integral membrane proteins (IMPs)
and the nuclear lamina, nucleoli, the nucleoskeleton, and a range of nuclear bodies
(Foster and Bridger 2005). All these structures interact with and anchor interphase
chromosomes. Misorganization or disruption of this nuclear architecture can lead to
problems in regulating normal chromosome behavior, producing compromised cells
with the possibility that diseases such as cancer or degenerative syndromes may arise.

Nuclear Structures
The most prominent subcompartment of the nucleus is the nucleolus. The nucleoli
are where ribosomal RNAs are synthesized and processed, thereby providing a site
for efficient assembly of ribosomal subunits. In humans the acrocentric chromosomes containing the ribosomal repeat genes are embedded in the nucleoli, providing a functional anchorage site for these genes and their chromosomes (Bridger
et al. 1998). tRNA genes are also clustered at the nucleoli (Boisvert et al. 2007;
Nemeth et al. 2010). Furthermore, other chromosomes that do not contain ribosomal DNA or tRNA genes are also associated with nucleoli (Bridger et al. 1998).

Interphase Chromosome Behavior in Normal and Diseased Cells

11

Fig. 2.1 Chromosome territories in interphase nuclei. Individual territories for human chromosome 10 (green) have been delineated using two-dimensional (2D) fluorescence in situ hybridization (FISH). (a) Normal immortalized human dermal fibroblast interphase nucleus. (b)
Immortalized HutchinsonGilford progeria syndrome fibroblast nucleus. (c) Nucleus from a transformed cell derived from a breast tumor. Note that the nuclei in (a) and (b) contain only two territories whereas (c) displays many territories of chromosome 10 that are derived from ploidy and
translocations

In genome-wide screens, two studies have revealed many sites throughout the
genome that are anchored at the nucleoli, including chromosome 17 (van
Koningsbruggen et al. 2010) and chromosome 19 (Nemeth et al. 2010). Both these
chromosomes have been found to be interiorly located and associated with nucleoli
in extracted nuclei (J. Bridger, unpublished data). These studies demonstrate that
the nucleolus is a major player in anchoring and organizing chromosome territories
in interphase nuclei.
The interior of a nucleus is thought to be more conducive to transcription of
active genes, whereas a correlation has been shown between gene repression and
positioning at the periphery (Zink et al. 2004). In yeast the nuclear periphery has
been shown to consist of two distinct compartments: a region permissive to transcription near the nuclear pore complexes, and a repressive region that contains foci
of silencing factors (Andrulis et al. 1998; Taddei et al. 2006). Components of the
nuclear periphery, such as nucleoporins and lamin proteins, are thought to interact
with repressors of transcription. For example, emerin interacts with the transcriptional repressors germ cell-less (GCL) and barrier to autointegration factor (BAF)
(Holaska et al. 2003), and the nuclear envelope protein LAP2 interacts with
HDAC3 to cause histone H4 deacetylation and gene repression (Somech et al.
2005). Genes can become anchored at the periphery of the cell, which affects their
local chromatin environment. For example, genes that become tethered to the
nucleoporin Nup2p are blocked from becoming heterochromatic and therefore
remain active, whereas tethering of telomeres to other nucleoporins results in gene
silencing (Ishii et al. 2002; Feuerbach et al. 2002).
The nuclear envelope is made up of the inner and outer nuclear membranes,
which house nuclear pore complexes, and the nuclear lamina. The inner nuclear
membrane, facing the nuclear interior, contains a large number of IMPs (TrinkleMulcahy and Lamond 2007; Gomez-Cavazos and Hetzer 2012). By proteomic analysis, at least 67 IMPs have been identified. The better known IMPs are lamin B

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receptor, lamin-associated polypeptides 1 and 2, emerin, MAN1, and nesprins

(Schirmer et al. 2003). Many of these IMPs have chromatin/DNA-binding capabilities and are believed to be involved in anchoring interphase chromosomes to
the nuclear periphery (Zuleger et al. 2011). Furthermore, the nuclear envelope
components can be very different in different tissue types (Korfali et al. 2012),
which could explain why different areas of the genome become localized to the
nuclear envelope in different cell types (Peric-Hupkes et al. 2010).
The nuclear lamina, found subjacent to the nuclear membrane, consists of type V
intermediate filament proteins nuclear lamins A, B, and C and is known as a structural scaffold under the nuclear envelope, which provides mechanical strength.
There are three mammalian lamin genes: LMNA, LMNB1, and LMNB2, encoding at
least six proteins. LMNA encodes four alternatively spliced mRNAs for lamin A,
A10, and C1 and C2 proteins, which are called A-type lamins. LMNB1 encodes
lamin B1, and the lamin B2 mRNA can be spliced to yield B2 or B3 proteins. The
presence of lamins A and C is limited to differentiated cells; however, lamins B1
and B2 are expressed in all cell types both in adults and in embryos. Furthermore,
expression of certain lamin proteins such as C2 is restricted to the testis and during
meiosis, whereas lamin B3 exists only in oocytes and spermatozoa (Rodrguez and
Eriksson 2010). Lamin proteins have DNA/chromatin-binding abilities but also
bind to a number of the IMPs of the nuclear membrane. Thus, there are a plethora
of sites at the nuclear periphery for interphase chromosomes to bind and be
anchored. A large study, in which the human genome was probed for lamin
B-binding sites, revealed 1,300 lamin-associated domains (LADs); many of these
LADs were found to be in gene-poor regions of the genome (Guelen et al. 2008).
Interactions with the nuclear lamina are associated with gene silencing and repression markers such as H3K4 dimethylation (Ferrai et al. 2010), increasing evidence
for the idea that the periphery of the nucleus is associated with gene repression.
Moreover, the disregulation of expression of both types of nuclear lamin has been
correlated with cancer and degenerative disease (Butin-Israeli et al. 2012), including neurological degeneration (Coffinier et al. 2011).
The movement of chromatin in the nuclei appears to be largely constrained and
thus reflects the physical attachment of chromatin to nuclear compartments, such as
the nucleolus, nuclear periphery, and nucleoskeleton. Individual chromosomes
occupy discrete compartments, and therefore distinct genomic regions localize to
specific subnuclear positions. From several studies, it is becoming evident that
nuclear position may have a crucial role for gene regulation. Moreover, it has been
shown that there is a strong correlation between transcriptionally silent, latereplicating chromatin and a nuclear peripheral localization in several model systems
(Boyle et al. 2001; Andrulis et al. 1998). Fluorescence recovery after photobleaching (FRAP) studies on mammalian cell nuclei indicate that in time periods of more
than 1 h, chromatin becomes immobile over distances greater than 0.4 m. Chubb
et al. demonstrated that nucleoli and the nuclear envelope constrain the motion of
interphase chromosomes that are located at these nuclear structures. In addition they
demonstrated that the mobility of chromatin not associated with nucleoli or the
nuclear periphery was much less constrained (Chubb et al. 2002).

Interphase Chromosome Behavior in Normal and Diseased Cells

13

Interphase Chromosome Positioning

We have been discussing how gene-poor regions of the genome are associated with
the nuclear periphery. These data come from sophisticated global screening experiments. This distribution of more inactive areas of the genome at the nuclear periphery fits with earlier studies whereby whole gene-poor chromosomes were found to
be located at the nuclear periphery. The first of these studies was performed by
Bickmore and colleagues and demonstrated the differential distribution in interphase nuclei of the similarly sized chromosomes 18 and 19 (Croft et al. 1999).
Chromosome 18, a gene-poor chromosome, was located at the nuclear periphery
whereas chromosome 19, a gene-rich chromosome, was located in the nuclear interior. This gene density-correlated chromosome positioning in interphase nuclei was
confirmed for all chromosomes in proliferating lymphoblastoid cells (Boyle et al.
2001). For human fibroblasts this gene density distribution is found in proliferating
cells and not in nonproliferating cells (Bridger et al. 2000; Meaburn et al. 2007a,
2008; Mehta et al. 2007, 2010). Nonproliferating cells display a size-correlated distribution with large chromosomes toward the nuclear periphery and smaller chromosomes in the nuclear interior. Thus, when doing chromosome positioning studies
it is critical to know whether the cells are proliferating. This point is especially
important when comparing transformed and immortalized cancer cells with primary
control cells, which will have a greater proportion of nonproliferating cells in the
culture or tissue section. We use immune detection of the proliferation marker
Ki-67, commonly used in neoplastic diagnostics (Kill 1996). The nuclei with very
bright staining are in the proliferative cell cycle, and negative nuclei or nuclei with
very dull staining are nonproliferating and are either quiescent or senescent. It is
important that the cells with very dull staining are not counted as positive because
this will lead to misinformation about proliferative status. In a primary culture of
fibroblasts the maximum number of proliferating cells is usually never more than
65 %, and this is for the youngest of cultures. Therefore, pKi67 is a very important
marker to use in chromosome positioning assays, but it must be analyzed correctly.
During the past decade there have been a number of studies that have compared
chromosome territory position between cancer cells and suitable control cells.
However, very few of these have taken into account proliferative status.
We have found that individual chromosome territories change location in the cell
nucleus when primary fibroblasts change proliferative status (Meaburn et al. 2007a;
Mehta et al. 2007, 2010), meaning that some specific chromosomes are relocated
whereas some stay where they are. Indeed, when cells are induced to become quiescent by serum starvation, interphase chromosomes either remain where they are,
such as chromosome X, or move from a peripheral location to a more interior location, such as chromosomes 13 and 18, or move toward the nuclear periphery, as, for
example, chromosome 10. We have shown that energy is required for the movement
of these chromosomes, and nuclear motor proteins actin and nuclear myosin I are
involved. Others have also found that nuclear motor proteins are involved in chromatin relocation in the nucleus (Chuang et al. 2006; Dundr et al. 2007; Ondrej et al.

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2007, 2008a, b). The idea that chromatin and chromosomes are translocated around
the nucleus by nuclear motor proteins is a relatively new area of study for nuclear
biologists, and as yet very little is known about the distribution and mode of action
of the nuclear motor proteins themselves. Our studies have shown that NM1b is
found throughout the nucleoplasm, with a concentration around the nuclear envelope and nucleoli in proliferating cells (Bridger and Mehta 2011; Mehta et al. 2010).
It is extremely likely, given the importance and reproducibility of chromosome and
gene positioning, that motor proteins involved in repositioning chromosomes and
chromosomal subregions could be altered in disease states and cause issues for gene
regulation. Indeed, we have observed in nonproliferating cells that NM1b distribution is very different, with large aggregates of the protein deep within the nucleoplasm. A similar distribution is apparent in cells derived from patients with the
premature aging disease called HutchinsonGilford progeria syndrome (HGPS)
(Mehta et al. 2011). Little or no research has been performed assessing nuclear
myosins in cancer; however, one study correlated the presence of nuclear myosin VI
with prognosis in renal cancer (Ronkainen et al. 2010) and another with nuclear
myosin 18b in ovarian cancer (Yanaihara et al. 2004).

The Link Between Interphase Chromosome Location

and Gene Expression
There is now evidence supporting the hypothesis that nuclear location of a chromosome and/or gene could play a role in regulating specific gene expression. For
example, when resting human lymphocytes are activated by phytohemagglutinin,
changes result in the intraorganization of chromosome territories, both in the degree
of intermingling between territories and in their volume. More importantly, however, the radial positioning of the chromosome territories is changed. This alteration
has been postulated to be a response to an altered transcriptional program (Branco
et al. 2008). Furthermore, during ex vivo stem cell differentiation into adipocytes,
the radial position of important genes involved in adipogenesis altered dramatically,
with genes that become switched on when moving from the nuclear periphery
toward the nuclear interior and back again when switched off. Control genes in this
system that were either on or off did not respond to the adipogenic growth factors
and did not change location (Szczerbal et al. 2009). In this differentiation system,
there was little whole chromosome movement, but genes were looped out from
chromosomes into the nuclear interior to associate with the nuclear structure SC35
speckles (Szczerbal and Bridger 2010).
Other studies have gone further, to identify where in the nucleus and to what
nuclear structures the genes are targeted. Genes have been found to relocate to
structures associated with active transcription and processing of RNA. Indeed, the
activation of gene loci can involve a repositioning of genes toward areas of the
nucleus where RNA polymerase II molecules aggregate into superstructures called
transcription factories (Osborne et al. 2007). Other studies have shown genes

Interphase Chromosome Behavior in Normal and Diseased Cells

15

becoming associated with other structures. For example, Dundr et al. inserted an
artificial U2 snRNA array into the genome of cells and demonstrated that the array
moved toward a stably positioned Cajal body for transcription through long-range
chromosomal relocation. This movement was inhibited by an actin inhibitor, implying the involvement of actin in interphase chromosome repositioning (Dundr et al.
2007). Other studies have shown genes increasingly associated with SC35 domains
upon upregulation (Brown et al. 2008; Szczerbal and Bridger 2010). It has also been
shown that repositioning of genes from the periphery to a more interior position can
correlate with inappropriate activation of that gene. The formation of chromatin
loops for expression from repressed chromatin territories has been suggested as a
mechanism of genome regulation, for example, for Hox gene activation (Chambeyron
and Bickmore 2004). Indeed, most excitingly recent 3C (Ferraiuolo et al. 2010) and
4C conformation capture experiments have shown that actively transcribing Hox
genes in a cluster are associated with a nuclear compartment for active transcription
and that the nontranscribing genes are all located at a region where gene silencing
occurs (Noordermeer et al. 2011). When the silenced genes become activated, they
then co-compartmentalize with the other active Hox genes. This strict cocompartmentalization of genes explains the strict co-linearity rules associated with
the Hox gene clusters where position in the cluster is correlated with the expression
zone down the developing embryo.
The three-dimensional (3D) structure of the chromosomes within the territories
also plays a major role in the control of gene expression. Regions of the chromosomes have been shown to interact with other regions of the same chromosome, in
cis. For example, the locus control region (LCR) of the -globin gene cluster acts as
an enhancer of the -globin genes, although it is more than 50 kb away. However,
the LCR has been shown to be in close physical proximity to an actively transcribed
HBB gene, suggesting a direct regulatory interaction (Carter et al. 2002; Tolhuis
et al. 2002). This looping in 3D forms an active chromatin hub (ACH) for control of
the expression of the -globin genes (de Laat and Grosveld 2003), which dynamically associate with the LCR (Gribnau et al. 1998). As T-helper cells differentiate
from nave, uncommitted CD4-positive T cells, they show a transcriptional switch.
Initially, the cells transcribe low levels of both Th1- and Th2-specific loci and regulators, but as they develop they become committed to either the Th1 or Th2 program
(for review, see Murphy and Reiner 2002). Once a lineage has been established, it is
retained as a heritable trait. This process of lineage commitment and differentiation
involves the physical repositioning of regulators of gene expression. For example, it
has been shown that during Th1 differentiation, the GATA-3 and c-maf loci, which
encode upstream regulators of Th2 cytokines, were progressively repositioned to
centromeric heterochromatin and/or the nuclear periphery and repressed (Hewitt
et al. 2004). These findings demonstrate another level of interphase chromosome
behavior on gene expression, that is, that the intraorganization of a chromosome
territory is also important (Fig. 2.2).
Noncoding RNAs (ncRNA) can control gene expression by establishing local
repressive regions. For example, the Air ncRNA sets up a local cloud of RNA that
accumulates at the promoter of the imprinted Slc22a3 gene and silences it by

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Fig. 2.2 Active genes can coassociate within the interphase nucleus. A montage panel of RNA
FISH experiments demonstrates that the Hbb-b1 gene loci (green) and the Hba gene loci (red) localize together more than 20 % of the time when actively transcribing. This interaction, shown in
embryonic day 14.5 mouse erythroblasts, occurs although they are located on different chromosomes and demonstrates the preferential interaction of coregulated genes within the nuclear volume

recruiting G9a, an H3K9 histone methyltransferase (Nagano et al. 2008). The

imprinted Kcnq1 locus is also regulated by a paternally expressed repressive
ncRNA, Kcnq1ot1, which regulates a domain of up to 750 kb. However, local activation of genes may be able to overcome the regional silencing effects of ncRNAs,
as Kcnq1 transitions from monoallelic to biallelic expression during the development of the heart, and there have recently been shown to be both tissue- and stagespecific chromatin loops between the Kcnq1 promoter and newly identified DNA
regulatory elements (Korostowski et al. 2011). The most notable example of
ncRNAs silencing genes is X-chromosome inactivation, where the ncRNA Xist
silences an entire chromosome. The Xist ncRNA covers the chromosome that is
going to inactivate and condenses into a smaller, compact structure, which is associated with the periphery (Clemson et al. 1996). As silencing is established, a repressive nuclear compartment forms that excludes RNA polymerase II and transcription
factors. Transcriptional repression follows the formation of this compartment, possibly as genes become physically pulled down into the repressive environment,
where they are inaccessible to the transcriptional machinery (Chaumeil et al. 2006).
The few genes that remain expressed, for example, those in the pseudoautosomal
region, loop out of the repressive compartment to be expressed (Splinter et al. 2011).

Interphase Chromosome Behavior in Normal and Diseased Cells

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Interphase Chromosome Behavior in HutchinsonGilford

Progeria Syndrome
HutchinsonGilford progeria syndrome (HGPS) is a severe premature aging disease
that affects children. First described by Jonathan Hutchinson and Hastings Gilford
in the 1800s, this disease is recognized by a group of characteristics indicative of
premature aging. The most common of these include alopecia (hair loss), failure to
thrive (short stature and low weight), lipodystrophy (loss of fat), scleroderma of the
skin, and increased visibility of blood vessels. Initially, children appear unaffected,
but symptoms usually present around 1 year of age, leading to a mean age of diagnosis of 2.9 years. Another main characteristic of HGPS is heart disease. Patients
suffer from atherosclerosis/hardening of the arteries, which is sometimes associated
with calcification. This change, in combination with loss of smooth muscle from
blood vessels, leads to an increased risk of heart attacks and stroke. These are the
main causes of death in this disease, with the average life expectancy of a HGPS
patient being 13.5 years (Hennekam 2006).
HGPS is an extremely rare disease with an incidence of approximately 1 in every
48 million live births. Of these cases approximately 80 % are caused by the same
de novo mutation in the LMNA gene (De Sandre-Giovannoli et al. 2003; Eriksson
et al. 2003). This gene encodes both A-type lamins, making HGPS part of a group
of diseases known as the laminopathies. The classic mutation found in the majority of HGPS patients is the G608G mutation, which is a silent mutation at the protein level. At the DNA level, however, it causes activation of a cryptic splice donor
site, which results in an interstitial deletion of 150 amino acids from exon 11. This
deletion gives rise to a truncated protein, with a 50-amino-acid deletion, called
Progerin (Eriksson et al. 2003). Pre-lamin A and progerin are subject to the same
posttranslational modifications. The region deleted in progerin contained an important cleavage site for the enzyme ZMPSTE24, which removes the farnesylated
N-terminus of the protein, freeing it from the membrane. Lacking the cleavage site,
progerin therefore remains bound to the nuclear membrane. Interestingly, homozygous mutations in the ZMPSTE24 gene have also been found to cause an atypical
form of HGPS. Progerin expression is thought to have a dominant negative effect on
cell function; it has been shown to cause thickening of the nuclear envelope as well
as nuclear shape abnormalities such as blebs and invaginations (Goldman et al.
2004; Bridger and Kill 2004).
The association of the nuclear lamina with the chromatin and chromosomes
increased interest in genome organization and chromosome/chromatin localization
in cases of disruption to the nuclear lamina through mutation in LMNA, such as that
seen in HGPS. Genome organization has been shown to be disrupted in a number of
cells with lamin A mutation or that lack lamin A completely (Galiova et al. 2008;
Shimi et al. 2008: Taimen et al. 2009). Further, three studies have revealed mispositioning of whole chromosome territories in cells with LMNA mutations (Meaburn
et al. 2007a; Mewborn et al. 2010; Mehta et al. 2011). Interestingly, using chromosome 10 positioning that had previously been shown to occupy different nuclear

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locations in proliferating, quiescent, and senescent cells (Mehta et al. 2007, 2010),
Mehta et al. (2011) revealed that HGPS cells had a quiescent-type distribution of
this chromosome in proliferating HGPS fibroblasts. Complete reorganization of the
genome was, however, not observed because the X chromosomes were found at the
nuclear periphery in both control and HGPS cells. This mislocalization of chromosomes could be restored to normal when the HGPS cells were treated with farnesyl
transferase inhibitors that prevent progerin from being farnesylated. A global
genome-wide study of the sequences associated with progerin at the nuclear periphery in mouse cells confirms that A-type lamins are involved in chromatin and
genome organization in nuclei. Kubben et al. (2012) show that in cells with progerin
some genes have been relocated away from the nuclear periphery, whereas others
have enhanced association.

Cancer
Cancer is a disease characterised by genomic instability, resulting in unlimited cell
replicative potential. Transformation is a multistep process usually encompassing
many genetic modifications including aneuploidy, copy number variants, gene
mutations, aberrant DNA methylation patterns, and chromosomal rearrangements.
The majority of these changes promote increased oncogenic transcription, which
stimulates proliferation and inhibits apoptosis.
With increased understanding of chromosome territories in the interphase
nucleus, much work has gone into understanding the differences that emerge in
neoplastic tissue compared with normal samples. The observed changes have been
both on the global scale, such as loss of heterochromatin, and at the gene scale, such
as the repositioning of tumor-associated genes in cancer formation (Zhu et al. 2011;
Meaburn and Misteli 2008).
The changes in the nuclear architecture of cancer cells are so robust they have
been used in tumor diagnosis for more than 140 years. Since the first patient biopsy
was examined in 1860, many advances have been made in understanding cancer.
However, diagnosis still relies heavily on the analysis of cell morphology. Specific
nuclear markers of cancer include changes in nuclear size and shape, nucleolus
alterations, changes in chromatin organization, aberrantly shaped nuclear lamina,
and alterations to promyelocytic leukemia (PML) bodies (Zink et al. 2004).
Common nuclear shape changes include indentations and folds that are indicative of
a wide variety of cancers. Nuclear morphological changes with specificity to certain
cancer types include grooves or long clefts in the nuclear surface, which are associated with the expression of the papillary thyroid oncogene expressed exclusively in
papillary thyroid carcinomas (Fischer et al. 1998). Enlarged nucleoli are associated
with several cancer types; however, inconspicuous nucleoli are almost exclusively
indicative of small-cell anaplastic lung carcinoma (Zink et al. 2004). Observed
changes to chromatin structures include changes to heterochromatic foci, which are
areas of the nucleus that contain highly compact chromatin structures usually

Interphase Chromosome Behavior in Normal and Diseased Cells

19

associated with gene silencing (Hahn et al. 2010). The changes to heterochromatic
foci include loss, asymmetry, coarse appearance, and spreading throughout the
nucleus (Zink et al. 2004). Several studies have identified silencing of tumor suppressor genes in cancer in parallel with changes to chromatin structure (Hahn et al.
2010). Tumor suppressor gene promoters showing heterochromatic markers such as
H3K9 trimethylation have been identified in many cancer types (Lakshmikuttyamma
et al. 2010). It is yet to be established whether changes in chromatin structure cause
silencing of tumor suppressor genes and thus drive cancer. One study conversely
found that knockdown mice lacking the tumor suppressor gene BRCA1 resulted in
changes to heterochromatin, including loss of foci from the nuclear periphery, leading to a more diffuse state of foci throughout the nucleus (Zhu et al. 2011). This
finding suggests some sort of positive feedback whereby tumor suppressors regulate
chromatin conformation; but once lost, aberrant chromatin changes promote
cancer.

Chromosome Positioning in Cancer

A vast number of diseases present with genetic defects that are often visible as chromosome rearrangements. The presence of chromosome abnormalities is a hallmark
for many forms of cancer. In many cases the specific association of certain chromosome aberrations and type of tumor are considered of diagnostic and prognostic
value.
Studies on interphase chromosome position in cancer cells were initiated by
work where HT1080 fibrosarcoma cell transformation was chemically reversed.
Acrocentric chromosomes that had been found through the nucleus were relocated
more centrally, which seems to result from their association with the nucleoli and
the coalescence of many smaller nucleoli to one prominent centrally located nucleolus after the treatment (Krystosek 1998). In a study from the Cremer laboratory, the
differential positioning of human chromosomes 18 and 19 was much less obvious in
colon adenocarcinoma cells, cervix carcinoma cells, and Hodgkin disease-derived
cells (Cremer et al. 2003). The nuclear locations of chromosomes 10, 18, and 19
were assessed in normal thyroid tissue and compared to adenomatous goiters, papillary carcinomas, and undifferentiated carcinomas. There was no difference in chromosome position in the normal and goiter tissue with chromosomes 10 and 18
positioned toward the nuclear periphery; and chromosome 19 in a central location.
However, in the papillary carcinoma tissue chromosome 19 was located centrally in
statistically fewer cells. Further, in undifferentiated carcinomas all the chromosomes assessed were mislocalized (Murata et al. 2007). In a breast cancer cell line
MCFCA1a differences in the distance between chromosomes 4 and 16 were found
when compared to the control cell line MCF10A (Marella et al. 2009). Wiech et al.
(2005) analyzed chromosome 8 positions in wax-embedded pancreatic cancer tissue samples. Radial distance indicated the repositioning of chromosome 8 to the
nuclear periphery, which matched roundness scores showing a change in the shape

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of the territory. A subsequent paper also noted a reduction of the roundness of chromosome 8 territories, suggesting a thinner, more elongated territory in pancreatic
carcinomas (Timme et al. 2011). The centromere and the gene encoding HER2 on
chromosome 17 were also shown to compact in neoplastic breast tissue, conferring
the repositioning of the centromere to a more internal location (Wiech et al. 2005).
A subsequent study by Wiech et al. (2009) reported repositioning of chromosome
18 during cell differentiation of nonneoplastic cervical squamous epithelium, showing a move toward the nuclear interior. This finding was in contrast to the observations in cervical squamous carcinomas that showed a repositioning of chromosome
18 toward the nuclear periphery (Wiech et al. 2009). This study also analyzed the
expression levels of BCL2, an inhibitor of apoptosis, which was shown to prolong
cell survival and found to be unregulated in 54 % of cervical cancers. A reduction
in BCL2 expression has been found in the terminally differentiated cells on the outer
layers of the cervical epithelium. In contrast, an increase in BCL2 expression was
found in the carcinomas, suggesting that relocation to the nuclear periphery
increases BCL2 transcription (Wiech et al. 2009).

Gene Repositioning in Interphase of Cancer Cells

A comprehensive study into the nuclear organization in breast cancer by Meaburn
et al. (2009) found that 8 of the 20 gene loci analyzed showed significant gene repositioning in cancer cells. All the gene loci studied have previously been implicated
in cancer, with the most frequently repositioned gene locus being HES5, a transcription repressor that regulates cell differentiation. As the majority of genes were not
repositioned, this finding suggests that the repositioning was gene specific rather
than global genome reorganization. It was also concluded that gene repositioning
was not associated with genome ploidy because the genes analyzed in this study had
no changes in copy number. It was also observed that some genes were only repositioned in certain cancer types, suggesting that some gene repositioning is cancer
type specific (Meaburn et al. 2009). This idea is supported by the findings of Wiech
et al. (2005), who identified BCL2 repositioning to the periphery in BCL2-positive
cervical squamous cell carcinomas but not in BCL2-negative cancer cells. By analyzing gene position in normal cells and cells from noncancerous disease, breast
hyperplasia or fibroadenoma, no significant difference was found (Meaburn et al.
2009). This result demonstrated that the rearrangements observed in cancer cells are
cancer specific and cannot be seen in noncancerous diseased cells. The identification of cancer-specific genes repositioned in several types of breast cancer could
prove a useful diagnostic tool. One problem this technique faces, however, is the
intermingling of normal and diseased cells, which reduces the statistical power. It
has been observed that the tissue directly adjacent to the cancerous tissue in patients
has a normal pattern of gene organization that matched with the tissue from normal
individuals (Meaburn et al. 2009). This finding is in agreement with previous reports

Interphase Chromosome Behavior in Normal and Diseased Cells

21

that the organization of genes does not differ between individuals, except in the case
of disease (Wiech et al. 2005). Once validated in a larger number of patient samples,
these problems should be overcome, yielding a useful diagnostic tool. Although the
study by Meaburn et al. (2009) failed to show a relationship between gene repositioning and transcription levels, other studies investigating this aspect have shown
that an altered positioning of specific genes in the nucleus is associated with altered
transcription levels (Wiech et al. 2009). This finding is in contrast with a previous
report that showed that gene repositioning occurs in early tumorigenesis and does
not affect transcription levels (Meaburn and Misteli 2008). More work is needed in
this area to understand why gene repositioning occurs, especially if it is not related
to gene function. Seminal studies aimed at understanding differentiation and maturation of the lymphoid lineage have analyzed the relationship between gene positioning and activity. One of these studies showed preferential localization to the
nuclear interior of the IGH and IGk loci during pro-B-cell lymphocyte development
(Kosak et al. 2002). This repositioning event coincided with transcription, and subsequent recombination of these loci is required for the production of unique antibodies. This finding led the authors to conclude that chromatin rearrangement is a
powerful mechanism for the control of transcription (Kosak et al. 2002). Further
studies will elucidate the role of gene positioning within the nuclear architecture as
an underlying condition for gene transcription and expression in cancer development and progression.

Formation of Chromosome Translocations in the Context

of Nuclear Organization
Cancer cells harbor a number of genetic abnormalities, of which chromosomal
translocations are well-studied examples, especially in leukemia and lymphoma.
The mechanisms of translocation formation are under study. Whether a multistep
process or a simultaneous occurrence of several events (Forment et al. 2012), we
expect an impact on genome organization and nuclear architecture. In any case, the
exchange of chromosomal fragments requires the formation of two or more doublestrand breaks (DSB). The incorrect repair of DSB leads to the fusion of nonhomologous chromosome ends, creating derivative chromosomes. The most error prone
pathway for the repair of DSB is nonhomologous end-joining, in which two chromosome ends in close proximity are religated. Within the nucleus there are errorfree repair pathways for DSB resolution, such as homologous recombination;
however, this process does require either a sister chromatid (post S-phase) or
homologous chromosome (Meaburn et al. 2007b). There is an ever-growing list of
cancer type-specific translocations, with the same rearrangement arising nonrandomly, and hence observed in the cancer cells of many individuals (Mitelman
Database of Chromosome Aberrations in Cancer, [Link]
Chromosomes/Mitelman). These recurrent translocations are useful diagnostic

22

G. Bourne et al.

tools and are often associated with clinical outcome. A classical example is the
Philadelphia chromosome derived from the t(9;22) and found in chronic myeloid
leukemia (CML) and in some cases of acute lymphoblastic leukemia (ALL)
(Goldman 2010). Another example is the t(12;21), found in approximately one third
of pediatric patients with ALL and associated with a relatively good prognosis
(Harrison et al. 2010). With increased understanding of the organization of the interphase nucleus, a number of studies have investigated the proximity of chromosomes
involved in common translocations as well as the 3D positioning of their derivatives
(Meaburn et al. 2007b) in certain cancers. There is evidence to support the hypothesis that translocations occur in interphase nuclei between chromosomes that
occupy similar nuclear space (Kozubek et al. 1999; Parada et al. 2002; Kuroda et al.
2004; Gandhi et al. 2012); this may also be true for intrachromosomal fusions and
genes that are at some distance linearly but may be placed together by chromosome
folding (Gandhi et al. 2006). Nuclear position was also identified as a factor that
contributed to translocation frequency, with peripherally located chromosomes such
as 4, 13, and 18 being involved in a higher than expected number of translocations
(Bickmore and Teague 2002). Later global screening studies have confirmed that
nuclear position is fundamental in the selection of translocation partners (Engreitz
et al. 2012; Roix et al. 2003), but transcriptional activity is also of fundamental
importance (Klein et al. 2011). A number of studies have been undertaken to understand the interaction of different chromosome regions that favor the exchange of
DNA fragments at the level of interphase nucleus, hence the formation of chromosome translocations (Branco and Pombo 2006; Murmann et al. 2005; Roix et al.
2003; Zhang et al. 2012). This point is supported by the finding that chromosome
territories do not have neat borders and that neighboring territories do intermingle
(Branco and Pombo 2006). It has been suggested that chromosomal translocations
are events whose frequency is correlated to the spatial proximity of the loci involved,
as described for some human lymphomas (Roix et al. 2003). One could speculate
that a similar location in the nuclear environment is sufficient to facilitate an encounter and an exchange of chromosome fragments, a phenomenon described as chromosome kissing (Cavalli 2007). Also, gene loci located on the periphery of
chromosome territories were found to be involved in more interchromosomal rearrangements than those deep within the territory (Gandhi et al. 2009). Internally
located loci were more frequently involved in intrachromosomal aberrations
(Gandhi et al. 2009).
Exposure to ionizing radiation results in DNA DSBs that permit nonhomologous
chromosomes in close proximity to combine, creating complex rearrangements
(Anderson et al. 2002). This rearrangement can cause several cancer types, in particular, radiation-induced thyroid tumors, such as papillary thyroid cancer. A common chromosome rearrangement observed in papillary thyroid cancer is the
intrachromosomal inversion on chromosome 10 that creates the fusion gene
RET/PTC1. Although the two genes involved in this inversion are on the same chromosome, they are 30 Mb apart. One study, however, showed that at least one copy
of each gene colocalized in 35 % of normal thyroid tissues compared with only 6 %

Interphase Chromosome Behavior in Normal and Diseased Cells

23

in mammary epithelial tissue (Nikiforova et al. 2000); this explains the tendency to
form inversions specifically in the thyroid. Another translocation that may arise
from exposure to ionizing radiation is the t(9;22)(q34;q11). This translocation gives
rise to the Philadelphia chromosome and the BCR-ABL fusion gene. The oncogenic
chimeric protein produced drives the formation of CML as well as some cases of
ALL. It has been shown that the BCR and ABL genes are found in distinct locations
in the interphase nucleus of healthy stimulated and nonstimulated lymphocytes
(Lukasova et al. 1997). In response to ionizing radiation, however, both genes were
shown to move to a more internal location, reducing the distance between them to
less than 1 m in 47.5 % of healthy donors (Lukasova et al. 1997; Kozubek et al.
1997). This finding suggests that rearrangement of chromatin in response to ionizing radiation brings into close proximity two genes known to be common translocation partners.
Regions from different chromosomes can also be brought into close proximity
by association with specific nuclear structures such as nucleoli (Sullivan et al.
2001). The acrocentric chromosomes (13, 14, 15, 21, and 22) of the human genome
all contain nucleolar organizer regions (NOR), which are composed of ribosomal
gene repeats. Nucleoli form around these NOR elements after mitosis, and during
cell-cycle progression the nucleoli fuse, creating fewer larger structures. However,
it should be noted that this process is very rarely observed in cancer (Morgan et al.
1987), whereas it is more likely to affect gamete formation and offspring as a result
of loss of genetic material. This process does, however, demonstrate that association
with nuclear elements can increase the occurrence of translocations. One example
specific to cancer is the translocation observed between the mouse chromosomes 12
and 15, which is present in 80 % of plasmacytomas (Osborne et al. 2007). The
breakpoints in this translocation involve the c-Myc gene and immunoglobulin heavy
chain locus (IgH). This translocation is mirrored in humans by that of t(8;14), which
encompasses the same genes and is found in Burkitts lymphoma as well as other
forms of lymphoid cancers (Haluska et al. 1987). These genes are found in close
proximity in only a third of human nuclei but are neighbors in mouse cells (Parada
et al. 2004; Roix et al. 2003). A study by Osborne et al. (2007) found that upon
activation both genes are recruited to the same transcription factories, increasing
their physical proximity. As proximity has been shown to be a key factor in translocations, this increases our understanding of why the t(12;15) is observed so frequently. In support of this realization, it was observed that c-Myc colocalized and
transcribed with IgH at the expected frequency to give rise to the observed level of
translocations (Osborne et al. 2007). The other translocation partners of c-Myc in
Burkitts lymphoma and plasmocytoma include IgK and IgL. The colocalization of
c-Myc to these genes was also analyzed and found to correlate with translocation
frequencies. Therefore, this research suggests a correlation between the number of
times genes come into close proximity and the likelihood of translocations (Osborne
et al. 2007).
Proximity is only one factor thought to affect translocation frequencies; other
factors to consider include chromosome size and gene density (Bickmore and

24

G. Bourne et al.

Teague 2002). One study found a correlation between chromosome size and translocation frequency in response to ionizing radiation (Cafourkova et al. 2001).
Another comprehensive study that analyzed more than 11,000 non-disease-causing
chromosome aberrations found that larger chromosomes were more frequently
involved in translocations; this could, however, be ascribed to increased opportunity
for translocations in large chromosomes because they are bigger targets. This study
also identified that translocations appear to occur less frequently in highly dense
regions of the genome (Bickmore and Teague 2002).

Repositioning of Genes Affected by Translocation Events

It has been postulated that, because of a translocation event, specific genes might
alter their position in the nucleus and therefore be more or less exposed to the transcription machinery. More precisely, certain genes could be activated or inactivated
on the basis of the new environment they inhabit. This change would happen when
two different regions characterized by different transcriptional activity become
positioned next to one another (as in the case of a reciprocal translocation), resulting
in an aberrant localization in the nucleus for one or both of the two regions. Studies
on both constitutional syndromes and cancer have focused on the localization of the
derivative chromosomes in the cell nucleus and also explored gene expression in the
context of the newly established nuclear architecture (Ballabio et al. 2009; Harewood
et al. 2010). A study on Ewing sarcoma cells has shown that the fusion genes derived
from the cancer-associated rearrangement t(11;22) assume an intermediate nuclear
position when compared to the wild type EWSR1 and FLI1 genes (Taslerova et al.
2003). Murmann and coworkers observed that the change in position of loci affected
by a translocation depends on the relative gene density of the 2-Mb window of the
region considered. The study of wild-type MLL and five of its translocation partners showed that the resulting fusion genes changed their nuclear location according to the reciprocal gene density of the region involved (Murmann et al. 2005).
More recently, a study on pediatric leukemia characterized by the presence of the
acquired t(7;12) translocation has shown that an overexpression of the HLXB9 gene
(on chromosome 7q36) corresponded to an altered nuclear position of the derivative chromosome carrying the HLXB9 gene itself (Ballabio et al. 2009). In this case,
the translocated HLXB9 gene localized more centrally than the wild-type allele.
A larger study on the constitutional balanced translocation t(11;22) has shown on a
larger scale that an altered spatial organization of the der(11) corresponds to an
alteration of the expression profile of genes localized on the der(11). In the same
study, chromosomes other than those involved in the rearrangement have also shown
an altered nuclear position and altered gene expression profiles (Harewood et al.
2010). This finding shows that the global nuclear architecture and the location of
various chromosomes are influenced by specific rearrangements. Altogether,
nuclear positioning plays a functional role in regulating gene expression.

Interphase Chromosome Behavior in Normal and Diseased Cells

25

Aneuploidy and DNA Copy Number Alterations

Gain and loss of genetic material is another common feature of cancer cells.
Microscopically, this defect can be visualized as complete loss or gain of entire chromosomes caused by missegregation during mitosis and resulting in aneuploidy. Other
imbalances are visible as loss or gain of certain chromosomal regions: these are known
as deletions, duplications, amplifications, or more generally as DNA copy number
alterations (CNA). These changes have an impact in diagnosis and are relevant at the
prognostic level. For example, extra copies of chromosome 3q define the difference
between cervical dysplasia and invasive cervical carcinoma resulting from human
papilloma virus (HPV) infection, whereas complete or partial loss of chromosomes 5
and 7 are the most commonly observed alterations in acute myeloid leukaemia (AML)
(Zhang et al. 2011). It is assumed that the presence of additional genetic material corresponds to increased expression levels of the overrepresented sequences. This
assumption is supported by a study on highly hyperdiploid pediatric ALL, showing
that the presence of additional chromosomal material corresponded to an increased
expression of the amplified loci (Gruszka-Westwood et al. 2004).
Very few studies have addressed the issue of chromosome organization in cases
of aneuploidy. Croft et al. (1999) did not see any repositioning of an extra chromosome 18 in Edward syndrome cells with a trisomy 18 (Croft et al. 1999). This finding is supported by that of Koutna et al. (2000), who investigated specific trisomic
loci within the HT-29 colon cancer cell line. They concluded that the location of the
third copy of a specific locus is not significantly relocated when compared to the
two loci present in a noncancerous tissue (Koutna et al. 2000). Although gene
amplification resulting from aneuploidy has the ability to drive cancer formation it
does not alter the organization of chromosome territories. Therefore, according to
these studies, tumorigenesis appears to be independent of chromosome position. In
another study, additional copies of chromosomes 7, 18, or 19 were artificially introduced in immortalized or cancer cell lines, and their position in the nucleus was
observed and correlated with altered expression levels. It was ascertained that the
presence of additional chromosomes increased transcription from the trisomic loci.
However, a shift in positioning was noted for chromosomes 18 and 19, but not for
chromosome 7. The authors proposed that positioning within the nucleus is determined by a unique chromosome-specific zip code that might be independent from
the transcriptional activity of the sequences that compose it (Sengupta et al. 2007).

Other Disease Situations

In 1988, Manuelidis and Borden published their seminal work demonstrating that
specific chromosomal domains were located to specific regions of the nuclei of
neurons and glial cells. In large neurons, probes delineating chromosomes 9, 1, and
Y were most commonly found adjacent to nucleoli. However, in astrocytes these

26

G. Bourne et al.

same regions were found at the nuclear membrane and not specifically associated
with nucleoli. These data indicate that nonrandom chromosome positioning is of
importance to the cell even in terminally differentiated cells such as nerve cells.
Manuelidis was the first to show in the human cortex the spatial repositioning of
chromosome in interphase nuclei in disease. She found that chromosome X had
become relocated from the nuclear edge to the nuclear interior in seizure foci in
epileptic patients. This study is significant because it links chromosome positioning
with ill health. In this study we do not know if the repositioning affects gene expression on the X chromosome. However, one of the master regulator genes for epilepsy
has been identified on the X chromosome (Stromme et al. 2002).
Very few studies have concerned chromosome and gene repositioning after an
infection. However, genes have been observed to relocate within cells of hosts that
are exposed to infectious agents. In Biomphalaria glabrata cells, the secondary host
organism of the human parasitic disease schistosomiasis, commonly known as bilharzia, specific genes involved in the infection become relocated within the interphase nuclei at the same time that quantitative polymerase chain reaction (PCR)
reveals that they are being expressed (Knight et al. 2011; Arican, Ittisprasert,
Bridger, and Knight, manuscript in preparation). One other study revealed chromosome 17 and not 18 changed nuclear location over time after an EpsteinBarr virus
(EBV) infection (Li et al. 2010).

Concluding Remarks
As more laboratories consider the 3D and 4D nuclear organization of the genome in
their studies on genome function, it is becoming clearer that chromosome position
and association with nuclear structure matter a great deal with respect to regulating
gene expression in healthy cells and affect the functioning of diseased cells when
misorganization of the chromosomes and genes is apparent. Furthermore, misorganization and misplacement of chromosomes and their gene loci may be responsible
for some disease situations.

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