Review articles

Concepts in nuclear architecture

Tom Misteli

Summary most central ones. I deliberately do not discuss any one

Genomes are defined by their primary sequence. The concept in detail, but aim to provide a broad overview of the
functional properties of genomes, however, are deter-
general ideas that form the framework for our understanding of
mined by far more complex mechanisms and depend on
multiple layers of regulatory control processes. A key nuclear function. For a detailed discourse of each topic, I refer
emerging contributor to genome function is the architec- the reader to several excellent recent review articles.
tural organization of the cell nucleus. The spatial and
temporal behavior of genomes and their regulatory The concept of nuclear compartments
proteins are now being recognized as important, yet still A central feature of the mammalian cell nucleus is its
poorly understood, control mechanisms in genome
morphological and functional heterogeneity generated by the
function. Combined cell biological, molecular and com-
putational analysis of architectural aspects of genome presence of distinct nuclear compartments.(1,2) A nuclear
function has added a further dimension to the investiga- compartment is defined as a macroscopic region within the
tion of some of the most fundamental cellular processes nucleus that is morphologically and/or functionally distinct
including transcription and maintenance of genome from its surrounding. Compartments are generally proteinac-
integrity. The complete elucidation of the contribution
eous nuclear bodies or chromatin domains (Fig. 1).
that nuclear architecture makes to gene expression will
be required to fully understand physiological processes
such as differentiation, development and disease at the Proteinaceous nuclear ‘bodies’
cellular level. Here I give an overview of some of the Nuclear compartments are membraneless suborganelles
emerging concepts in the study of in vivo genome characterized by a distinct set of resident proteins. The most
organization and function. BioEssays 27:477–487, prominent nuclear bodies include the nucleolus, which is the
2005. Published 2005 Wiley Periodicals, Inc.{
site of transcription and processing of ribosomal RNA, the
splicing factor compartments, which act as a storage/
Introduction
assembly site for spliceosomal components, the Cajal body,
Genomes function in the context of nuclear architecture.
which is the proposed site of snRNP assembly and the PML
Recent work has lead to the realization that the mammalian
body, which is of unknown function.(1) In addition to these
cell nucleus is a spatially and functionally compartmentalized
structures, many additional nuclear bodies characterized by
organelle containing numerous proteinaceous nuclear bodies
the presence of one or multiple nuclear proteins have been
as well as non-randomly positioned genome domains. The
described in the literature.(2) The function of many of these
spatial and temporal aspects of genome organization have
bodies remains elusive. However, conceptually at least three
likely functional consequences as indicated by the fact that
models must be considered for the function of a nuclear body.
changes in nuclear architecture are amongst the most dra-
First, the body may not be of functional relevance. The obser-
matic hallmarks of development and differentiation processes
ved morphological bodies may simply be a consequence of
and defects in architectural elements of the cell nucleus are
aggregation of excess protein that is not used (Fig. 1). The
now known to be responsible for several human diseases.
observation that overexpression of some nuclear proteins can
The key question of how genome function is integrated into
result in the formation of structures that appear by fluores-
the architectural framework of the cell nucleus and how
cence microscopy indistinguishable from nuclear bodies and
structural elements of the nucleus affect nuclear processes
the fact that in some cases overexpression of nuclear proteins
including gene expression, DNA repair and genome stability
results in the de novo formation of nuclear bodies are
are critical to our understanding of genome function. The
consistent with this notion.(3) Second, a nuclear body may be
intense study of nuclear architecture has lead to the
a site of particular nuclear activities. This model clearly applies
emergence of several general concepts, many of them
to the nucleolus, which contains all ribosomal genes and is the
surprising and provocative. I give here a brief overview of the
site of the vast majority of processing of ribosomal RNA.(4)
Similarly, recent work has suggested that CBs may be sites of
assembly of snRNP particles or, alternatively, it has been
National Cancer Institute, NIH, Bethesda, MD 20892.
E-mail: mistelit@mail.nih.gov
suggested that they are sites of assembly of transcription
DOI 10.1002/bies.20226 complexes.(5–7) A third model suggests that a nuclear body is a
Published online in Wiley InterScience (www.interscience.wiley.com). site of inactivity and may serve as storage site to provide
components to its immediate surroundings. This model has

BioEssays 27:477–487, Published 2005 Wiley Periodicals, Inc. BioEssays 27.5 477
{
This article is a US government work and, as such, is in the public
domain in the United States of America.
Review articles

Figure 1. Compartmentalization in the mamma-

lian nucleus. The nucleus contains proteinaceous
nuclear bodies, chromatin domains including
heterochromatin (dark grey) and euchromatin
(light grey) and chromosome territories. Nuclear
bodies can either be non-specific aggregates, sites
of nuclear processes (rRNA transcription in the
nucleolus; green) or sites of inaction (storage of
splicing components in splicing factor compart-
ments; red).

been applied to the splicing factor compartments.(8,9) These chromatin morphology and function is an oversimplification. A
compartments are enriched in pre-mRNA splicing factors but, recent study by Bickmore and colleagues has directly compar-
enigmatically, were found not to be sites of pre-mRNA splicing. ed the relationship of chromatin structure with gene activity on
However, upon activation of genes in the vicinity of these a genome-wide scale.(12) Using biochemically defined probes,
compartments, splicing factors are recruited from the com- the distribution of ‘open’, ‘closed’ and ‘bulk’ chromatin were
partments to the activated sites of transcription, suggesting mapped on intact chromosomes and onto DNA chips. Rather
that the compartment serves as a reservoir of factors without than finding a strong correlation between chromatin structure
being an active site of splicing.(10) In this function, compart- and gene activity, a link between chromatin morphology and
ments might contribute to regulate the concentration of gene density was found with gene-rich regions being present
available factors in the nucleoplasm and at their actual sites in open chromatin regions and gene-poor regions in more-
of action.(8) condensed domains, regardless of their activity status. It thus
The absence of membranes provokes the question of how appears that the view of euchromatin as regions of genome
nuclear compartments are formed and maintained. One activity and heterochromatin as regions of genome inactivity is
possibility is that the compartments contain structural proteins oversimplistic.(12)
that serve as a stable scaffold around which a nuclear body is Heterochromatin and euchromatin can also, roughly, be
formed. However, no skeletal proteins have been identified for defined molecularly based on differential post-translational
any of the known nuclear bodies. An alternative possibility is modifications of core histones.(13) Euchromatin is generally
that nuclear bodies are largely the results of the sum of many, enriched in hyperacetylated histones H3 and H4, whereas
likely transient and non-specific, interactions amongst its heterochromatin is characterized by hypoacetylation and tri-
resident proteins. In support of such a self-organizing model, methylation of histone H3 on lysine 9. The latter modification is
the potential for self-interaction has been reported for marker thought to serve as a mark for heterochromatin and as a
proteins of virtually all nuclear bodies.(1,11) binding site for structural heterochromatin proteins, which
likely contribute to maintaining the highly condensed state of
Chromatin domains chromatin. How precisely the morphological, biochemical and
In addition to proteinaceous bodies, large-scale chromatin molecular definitions of chromatin domains correspond to the
domains exist within the nucleus. The major chromatin functional status of the various chromatin regions is an issue of
domains are morphologically defined as euchromatin consist- current investigation.
ing of less-condensed genome regions, whereas heterochro-
matin contains more highly condensed regions (Fig. 1). The concept of chromosome territories
Euchromatin is generally considered to be the site of ‘open’, Chromosomes exist in the nucleus in the form of chromosome
presumably transcriptionally active, chromatin regions and territories.(14,15) This definition denotes the fact that the
heterochromatin is often thought of enriched in inactive and genetic material of each chromosome is not widely distributed
silenced genome regions. While the two types of chromatin throughout the nucleus but occupies a spatially defined
can easily be defined based on their morphology, it is subvolume (Fig. 1). The existence of chromosome territories
becoming increasingly clear that the correlation of large-scale was originally demonstrated by inducing DNA damage in a

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defined region of the nucleus using a microlaser and analysis in its arrangement of the genome, of nuclear compartments
of the damaged chromosomes in the subsequent meta- and of nuclear proteins. The non-random nature of nuclear
phase.(16) These ingenious experiments revealed that only a organization is one of the most striking and puzzling features of
small subset of chromosomes was damaged and that the the mammalian cell nucleus and the functional significance of
number of chromosomes was dependent on the size of the non-random nuclear organization is one of the current frontiers
irradiated area. These findings were most consistent with a in the field.
model in which chromosomes are arranged in spatially limited,
well-defined, nuclear subvolumes. This conclusion was later Genomes in three dimensions
directly confirmed by fluorescence in situ hybridization using Genomes are non-randomly arranged in three-dimensional
probes specific for entire chromosomes, which allowed the space.(14,15,19) A convenient, although highly simplistic,
visualization of chromosome territories in situ.(17) indicator of the position of a chromosome or a locus is the
Despite the fact that the outlines of the chromosome radial position, i.e. its location relative to the nuclear center or
territories are spatially well defined their internal structure is the periphery (Fig. 2A). Amongst many other examples, in
still ambiguous.(18) Initially thought of as solid domains with the lymphocytes and fibroblasts, the radial position has been
active transcribed genes at the surface and exposed to correlated with a chromosome’s gene density, with gene-rich
regulatory factors that might be present in canal-like structures chromosomes positioned preferentially towards the center of
between chromosome territories, it is now clear that chromo- the nucleus, and gene-poor chromosomes located towards
some territories are not solid structures, but rather are the periphery.(19–21) In other studies in fibroblasts, radial gene
permeated by nucleoplasmic channels of various sizes.(14) positioning has been correlated with chromosome size rather
This creates a porous entity with a highly convoluted and than gene density.(22) The non-random arrangement of
enlarged surface area. This structural property likely facilitates chromosomes is also reflected in the positioning of chromo-
access of regulatory factors to sequences buried within the somes relative to each other (Fig. 2A). In mouse lymphocytes,
chromosome territory. Chromosome territories are not uniform for example, single homologues of chromosomes 12, 14 and
in their structure and appearance and likely contain loops of 15 frequently form a non-random cluster.(23) This cluster only
varying sizes leading to intermingling between neighboring contains one of the two homologues with the others apparently
territories.(18) As a corollary of the discrete nature of chromo- randomly distributed throughout the nuclear volume. Interest-
some territories, the possibility arises that chromosomes are ingly, tetraploid cells frequently contain two such clusters,
arranged in particular patterns within the nuclear space.(15) suggesting that distinct rules of positioning apply to chromo-
some homologues.(23)
The concept of non-random nuclear organization The arrangement of chromosomes within the nucleus
One of the major emerging concepts in the study of nuclear appears to be tissue-specific.(20,24,25) A systematic study of
architecture is the non-random organization of the nuclear multiple chromosomes in several primary tissues has shown
space. The mammalian cell nucleus is highly non-random both that both the radial as well as the relative positioning of

Figure 2. Non-random positioning of genomes. A: Gene loci and chromosomes are non-randomly positioned radially relative to the
nuclear center and relative to each other. B: Tethering of gene loci (red, green) via boundary elements (purple) to the nuclear periphery or to
intranuclear compartments contributes to their regulation and reduces their dynamic mobility. C: Gene loci, regardless of their
transcriptional activity, exist in ‘open’ chromatin (yellow). When potentiated (green), loci may be displaced from their chromosome territory
and explore the nuclear space in search of functionally equivalent regions (grey). This region may either be a silencing environment such as
heterochromatin or an active environment such as a transcription factory. Association with the particular enviroment may results in changes
in the functional status of the gene (red).

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chromosomes relative to each other differs amongst tis- Possibly the functionally most important type of positioning
sues.(24) Statistical comparison of distribution patterns further is the location of a gene relative to chromatin domains,
suggests that cell types, which share differentiation pathways, particularly transcriptionally repressed heterochromatin do-
such as lymphocytes and myeloblasts, exhibit a more similar mains. The classic example of positioning-induced silencing is
three-dimensional organization of genomes than unrelated the phenomenon of position effect variegation (PEV). In PEV, a
cell types.(24) While these observations suggest a role for locus becomes permanently silenced due to physical place-
differentiation in determining positioning patterns, the exact ment into a heterochromatic genome neighborhood. In the
mechanisms that give rise to non-random chromosome case of the well-studied brown locus in Drosophila insertion of
positioning are unknown. a heterochromatin block near the locus results in its physical
An underlying concept in understanding three-dimensional positioning near a heterochromatin region and its silencing
genome organization is the probabilistic nature of positioning in trans via contact with the existing heterochromatin domain in
patterns. The radial or relative position of a chromosome is a sequence-independent manner.(32) Thus, positioning rela-
always a reflection of its preferred position, but does not tive to heterochromatin domain can clearly have a regulatory
indicate that a given chromosome is found in one particular effect. This effect is most likely not limited to artificial experi-
place in all cells of a population. Thus, while it should be mental systems, since numerous examples of correlations
possible to generate probability maps of how genomes are between proximal positioning of a locus and its silencing have
spatially organized, it will not be possible to define the position been reported in differentiation systems.(33) Having said that,
of a genome region in absolute terms. association with a heterochromatin region does not necessa-
rily confer transcriptional silencing, since some loci, such as
Gene positioning the l-5 locus in B-cells, retain their activity despite positioning
Sine chromosomes are non-randomly arranged in the within heterochromatin regions and a significant fraction of
nucleus, it is not surprising that the gene loci that they harbor genes are found in heterochromatin in Drosophila.(34)
are also found in non-random patterns. How the position of a It is not surprising to find that gene loci generally localize
locus relative to the nuclear periphery or relative to nuclear within the chromosome territory on which they localize.
landmarks, such as chromatin domains or the chromosome However, this is not always the case and both the dissocia-
territory, affects its function is one of the key questions in tion from territories as well as the preferential positioning of
understanding the contribution of spatial positioning to genome regions at the surface of territories has been sug-
genome function. gested to contribute to proper gene function(35,36) (Fig. 2C).
Although strong preferential positioning relative to the In human and mouse cells, several genome regions have
nuclear center has been observed for many analyzed genes, been characterized that are expelled from their chromosome
this positioning is likely not critical for function, since a territory and loop several micrometers away from the main
particular gene, regardless of whether it is active or inactive, body of the chromosome.(37–39) All of these domains contain
can be found at virtually any position in the nucleus within a cell gene clusters and are highly transcribed. Looping likely
population.(19,26 –28) It seems more likely that the non-random contributes to gene regulation as suggested by analysis of
positioning observed for loci is largely the consequence of the correlation between loop formation and gene activity
the positioning of the chromosomes on which the gene is during differentiation processes. In erythroid cells, the b-globin
found. On the other hand, changes in the functional status of locus loops out from the chromosome territory before it is
several genes have been reported to be paralleled by transcriptionally active, but remains associated with the
repositioning. For example, in T-cell differentiation, the CD4 chromosome when its regulatory elements are mutated.(35,40)
locus moves towards the periphery as it becomes inactivated Similarly, in differentiating ES-cells, the Hox1 and Hox 9 genes
during selection for CD8þ cells.(27) However, repositioning is dissociate from their territories in parallel with their activity.(41)
clearly not a pre-requisite for changes in function since, in the While the former example suggests looping as a consequence
same cells, the CD8 locus does not alter its intermediate radial of potentiation of the gene, the latter points towards a correla-
position regardless of its functional status.(27) tion with gene activity per se (Fig. 2C). Whether these exam-
More important than radial positioning might be tethering of ples represent different paradigms of positioning and how
gene loci to nuclear landmarks (Fig. 2B). In yeast, association common the different behaviors are remains to be established.
of gene loci with the nuclear periphery occurs in response to Genome regions may also be non-randomly positioned
both gene activation and gene inactivation depending on the relative to each other. The best example of spatial clustering of
gene.(29) Furthermore, in yeast and Drosophila, boundary genes is the ribosomal genes, which congregate non-
elements that functionally insulate neighboring genome randomly in the nucleolus. A typical mammalian nucleolus
regions appear to act by tethering the affected regions to a contains 50–100 active ribosomal genes located on 2–4
nuclear structure such as the nuclear periphery or the distinct chromosomes.(4) While it seems clear that the
nucleolus(30,31) (Fig. 2B). clustering of ribosomal genes enhances the efficiency of

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rRNA transcription and processing, it is not known what the nucleolus, where ribosomal genes are transcribed and the
mechanisms are that lead to the clustering of the chromo- resulting rRNAs processed.(4) The nucleolus invariably as-
somes. Similar clustering has recently been observed for sociates with the subset of chromosomes containing the
tRNA gene in S. cerevisiae, where more than 50 genes located tandemly repeated rDNA clusters. The association is driven by
on all 16 chromosomes cluster near the nucleolus.(42) Initial the transcriptional activity of the ribosomal genes since
evidence for spatial clustering of RNA pol II genes in introduction of rDNA genes on non-integrating plasmids leads
mammalian cells has recently also been reported.(40) Osborne to the formation of mini-nucleoli and inhibition of the ribosomal
et al., have found that two genes located 25 Mb apart on DNA-specific RNA polymerase I results in disassembly of
chromosome 7 pair in three-dimensional space to generally nucleoli.(4) Furthermore, the nucleolus disassembles during
share a common transcription domain. This observation mitosis when rDNA genes are silenced, but rapidly reforms
suggests that spatial gene clusters might be more prevalent upon re-imitation of ribosomal gene activity in late telophase.
than previously expected. Analogous to the situation of the nucleolus, Cajal bodies
The positioning of genome regions, be it chromosomes, appear to associate with relatively high frequency with histone
genome regions or single genes, might not only be functionally and U2 snRNA gene clusters and introduction of U2 snRNA
relevant for gene expression, but especially for cellular minigenes promotes the association of Cajal Bodies.(48–50)
processes that involve the physical interaction of genome These observations on nucleoli and Cajal bodies strongly
regions such as formation of translocations or recombina- suggest that the non-random association of these nuclear
tion.(43) In support of an important role of non-random compartments is a direct consequence of the transcriptional
positioning is the fact that strong correlations have been status of the target genes.
detected between the spatial proximity of chromosomes or Other nuclear bodies have been found in non-random
genes and their probability of undergoing translocations or proximity with genome regions, but it is not clear whether this
recombination. In human cells, the translocation partners for association is driven by specific genes or by broader genome
several lymphomas have been found in closer spatial proximity regions. For example, the OPT-domain (Oct1/ PTB/transcrip-
than would be expected based on their random distribu- tion) has been mapped to preferentially associate with a
30
tion.(26–46) Similarly, in yeast, the preferred recombination Mb region on human chromosome 6p21.(51) A particularly
partners involved in preferential donor selection during intriguing association is that of the heat-shock granules. These
mating-type switching are on average in closer spatial structures form in human cells upon heat shock. They
proximity than unrelated loci.(47) These observations suggest preferentially form at a satellite repeat region on chromosome
that the non-random spatial positioning of genome regions in 9 and, remarkably, concomitant with heat shock this normally
the interphase nucleus may contribute to determining what silent region becomes transcriptionally active and generates
genome region interact with what other regions.(15) an apparently non-coding RNA of unknown function.(52,53) In
Taken together, the role of positioning in gene function is still this case, it is not clear whether formation of the heat-shock
unclear. A major impediment towards deducing general rules granule drives transcription from this site or whether it
for how positioning affects gene function has been the represents the consequence of transcriptional activation of
limitation of most studies to the analysis of single genes the satellite region.
resulting in largely anecdotal reports of correlation between Several nuclear structures associate non-randomly,
positioning and function. More systematic analysis of multiple, although more generally, with active genome regions. PML
functionally related genes using high-throughput imaging bodies are preferentially found near active genes and splicing
methods and pattern recognition tools will likely resolve this factor compartments containing pre-mRNA splicing factors
issue in the future. In addition, the mining of whole genome often associate with gene-dense R-bands.(54,55) While the
sequence information to understand how the primary genome association of PML bodies with active genes does not appear
sequence affects higher order organization of chromatin and to be functionally important since depletion of PML has no
its environment and to ask whether higher order structure can affect on the association, it seems highly likely that the physical
be predicted from sequence information will be a necessary proximity of splicing factor compartments facilitates the
and promising, albeit challenging, approach to understand processing of transcription generated from the gene-dense
spatial genome organization and its link to function. R-bands. It thus appears that the non-random association of a
nuclear body with a genome region is generally the conse-
Positioning of nuclear bodies quence of the region’s activity.
Not only are genomes non-random arranged in vivo, but
increasing evidence also suggests that non-random position- The concept of nuclear dynamics
ing of nuclear bodies relative to specific genome regions is the The cell nucleus has recently emerged as a highly dynamic
norm rather than the exception. The clearest example of non- organelle and it is now clear that virtually all aspects of nuclear
random positioning of a nuclear body to gene loci is the function and organization are dynamic.(56) Photobleaching

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experiments have revealed that proteins roam the nuclear interact with their ‘home’ compartment where they are
space in an energy-independent manner in search for high- captured by higher-affinity interactions than they encounter
affinity binding sites.(57) This ability of molecules to freely in the rest of the nucleus.(56) This interpretation is consistent
diffuse through the nuclear space appears particularly with the discovery that many resident proteins of nuclear
important for chromatin-binding proteins.(58–61) These factors bodies frequently visit other nuclear structures.(67) Thus, the
have been shown to undergo highly transient binding interac- overall stable nuclear suborganelles are generated from
tions with chromatin both at their specific as well as their non- dynamic components.
specific binding sites. Estimated residence times for proteins Not only proteins and protein complexes exhibit dynamic
on chromatin, including structural proteins of euchromatin and motion in vivo, but nuclear bodies themselves are dynamic.
heterochromatin, are typically on the order of 2–30 s and the Although the nucleolus is positionally very stable and does not
time between binding events is on the order of 50–200 ms.(58) change its location over short periods of time, most other
The latter value indicates that, once dissociated from a binding nuclear bodies do. Cajal bodies undergo saltatory motion and
site, a chromatin protein is rapidly captured by a non-specific or PML bodies have been reported to be able to move in linear
a specific binding site in its vicinity. An implication of this trajectories through the nucleus.(68,72) Both of these move-
behavior is that at any given time a large fraction of any ments are sensitive to ATP levels, but it is difficult to ascertain
chromatin protein is bound, most frequently to a non-specific that this effect reflects an energy dependence of the motion
site, and that only a small fraction of the protein is unbound.(58) itself or is an indirect effect caused by the perturbation of large-
The combined transient interaction and the ability of proteins to scale chromatin structure due to ATP depletion.(69,71,72) What
diffuse rapidly suggest that chromatin proteins scan the the functional significance of nuclear body movement might be
genome for appropriate high-affinity binding sites by a three- is unclear, but it has been suggested that, similar to the
dimensional hopping mechanism.(58) situation for chromatin-binding proteins, the movement of
The molecular basis for the dynamic nature of protein– nuclear bodies allows them to scan the genome for preferential
chromatin interactions is unclear. Evidence from the analysis interaction sites.(69) This idea is consistent with the observed
of dynamic binding of steroid receptors in permeabilized cells non-random association of nuclear bodies with particular
suggests that the rapid turnover of steroid receptors is genome regions.
promoted by chaperone activities that either directly act on Gene loci themselves can undergo dynamic motion as well.
the receptors to remove them or indirectly cause their In vivo tracking of fluorescently tagged loci demonstrates that
dissociation by acting on some of their interaction partners.(62) they undergo significant constraint diffusional motion.(73–75) In
In addition, evidence from in vitro reconstitution and ultra-fast mammalian cells, a locus may explore its immediate environ-
crosslinking experiments has also suggested that the resi- ment by local motion within a subvolume of typically about
dence time of chromatin proteins may be related to chromatin 1–2 mm in diameter.(75) Remarkably, the absolute range of
remodeling, which might periodically destabilize the weak motion of a given locus is similar in mammalian cells and
binding of chromatin proteins.(63) In vivo observations using yeast.(74) A locus can deviate from its average position by
inhibitors have pointed towards a direct or indirect role for about 1 mm over the period of several minutes. However, in
proteasome activity in the dynamic exchange of chromatin absolute terms, this motion has very distinct significance in
proteins.(60,64) Given the large variety of chromatin-binding mammals and in yeast, since in mammals this motion
proteins, it seems likely that the mechanisms of exchange will corresponds to sampling of a volume roughly equivalent to a
greatly vary between different proteins. Regardless of the single chromosome, whereas in yeast this represents an
detailed molecular mechanisms for the transient protein– exploration of the entire cell nucleus. The motion of a locus
chromatin interactions, the functional significance of transient may be reduced by tethering to the nuclear periphery or
binding is likely the ability of these proteins to undergo multiple, intranuclear structures such as nucleoli.(75) This is particularly
rapidly changing interactions with partners on chromatin to evident by the immobilization at the nuclear envelope of
generate combinatorial protein complexes on chromatin.(56,58) telomeres in yeast.(74) The measured diffusion coefficients of
This property likely allows cells to alter their transcriptional the motion of a locus are orders of magnitude lower than those
output rapidly in response to stimuli and thus provides a of proteins and thus a locus largely appears immobile relative
molecular basis for the high transcriptional plasticity inherent to diffusing proteins. Whether the diffusional mobility of
to most cellular systems. genome loci has any functional significance is unclear.
Photobleaching experiments have also uncovered that
proteinaceous nuclear bodies including the nucleolus are The concept of nuclear scaffolds
generated from rapidly exchanging molecular compo- The complex spatial organization of the nucleus has given rise
nents.(57,65,66) Similar to the situation for chromatin proteins, to the tempting concept of a structural framework in the form of
it seems now most likely that ‘resident’ nuclear body proteins, a karyoskeleton or matrix that might contribute to spatially
diffuse relatively freely throughout the nucleoplasm until they organize the multitude of nuclear compartments.(76,77) The

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molecular identify of such a structural scaffold remains elusive. proteins, which compete with each other for binding sites in a
Several interesting candidates have been identified. In dynamic interaction network.(88) These dynamic, largely
Drosophila, the non-chromosomal EAST protein has been random interactions amongst proteins are consistent with
suggested to be part, or at least control, a putative nuclear the recent realization that gene expression events are highly
endoskeleton, since overexpression of the protein causes stochastic.(89,90)
expansion of the extrachromosomal space and the nuclear The principle of self-organization is also consistent with the
volume.(78) In human thymocytes, the SATB1 protein appears observed probabilistic nature of chromosome positioning and
to form a cage-like network of proteinaceous filaments to which can also account for the non-random positioning of genes
genes can be attached.(79) The association of several relative to nuclear landmarks. In this view, positioning may be
thymocyte-specific genes appears to correlate with their the result of the mutual interplay between the functional status
activity suggesting that association with this scaffold has a of a locus and the nuclear domain with which it interacts
regulatory function. (Fig. 2C). A plausible scenario is that a gene locus becomes
An obvious candidate for a scaffold component are the well- potentiated by specific local events such as binding of par-
characterized intermediate-filament-type lamin proteins, ticular regulators or histone modifications. The potentiated
which have long been known to form a structural meshwork locus may then be incorporated into a more ‘open‘ chromatin
underlying the nuclear envelope refereed to as the nuclear loop, which, in the extreme case, may become expelled from
lamina.(80) Recent observations strongly suggest that lamins the chromosome territory. The more flexible loop is now free
A, B and C are also present in the interior of the nucleus and are to associate with functionally distinct subregions of the nucleus
functionally critical for transcription and replication.(80) It is and may become preferentially associated with domains that
unclear what their contribution to the organization of nuclear are of functional equivalence. The interaction of the locus with
structures is at present, but at least lamins A and C are not these domains then in turn may stabilize its functional status,
essential for the formation of splicing factor compartments and resulting in a non-random preferential positioning.(43)
other nuclear bodies.(81,82) A further prominent candidate for a The auto-reinforcing behavior of self-organizing systems
structural component of a karyoskeleton is actin. It is now may contribute greatly to the overall stability of nuclear
established that monomeric actin is present in the nucleus and structure and the functional status of the genome but, at the
appears to be functionally relevant in transcription complex same time, the transient nature of virtually all protein–protein
formation; whether actin filaments exist in the nucleus is not and protein–chromatin interactions may also poise the system
known.(83) for rapid change in response to external stimuli. Thus, the
dynamic, self-organized, nature of nuclear organization is a
The concept of nuclear self-organization fundamental, functionally essential property of the cell.
The difficulties in unambiguously defining a static nuclear Self-organization models are notoriously difficult to experi-
skeleton and the observed highly dynamic nature of nuclear mentally test or falsify. While it is hard to imagine a single key
architecture has lead to the consideration of suggestions that experiment to prove that the nucleus is a self-organizing
the nucleus may be a self-organizing entity.(84,85) In this system, combining information from experimental and com-
model, the complex morphological appearance is a reflection putational approaches will likely allow critical probing of this
of the transcriptional activity of the genome and the sum proposal. Biochemical experiments will tell us whether a
of all molecular interactions amongst nuclear components. nucleus is able to self-organize into a functional entity from its
Although this provocative model is speculative at present, components as would be predicted. Simple nuclear re-
virtually all observations on nuclear organization and assembly systems capable of transport across the membrane
dynamics are consistent with it. Central to self-organizing and transcription of defined DNA have been used for years.(91)
systems is the high dynamic content and a relative promiscuity Furthermore, it has been demonstrated that nucleoli and PML
of interactions amongst components. The recent observations bodies dispersed by incubation in a low monovalent cation
of the dynamics of numerous nuclear proteins in living cells environment can reassemble and resume their cellular
clearly supports both of these premises. As discussed above, functionality by simple addition of inert macromolecules,
proteins can rapidly roam the nuclear space, the resident suggesting that both the structural and functional integrity of
components of subcompartments are dynamically exchanged nuclear compartments depends strongly on molecular crowd-
and many chromatin proteins undergo extensive non-specific ing events as would be expected for a self-organizing
transient interactions with chromatin and with nuclear system.(92) In addition to direct experimental interrogation,
bodies.(56) Consistent with a self-organization model, the the concept of self-organization will also be tested by the
major proteins that confer the structural identity of hetero- converging efforts of system biology approaches and compu-
chromatin exchange and stochastically find their specific tational modeling methods, which are now increasingly applied
binding site.(86,87) Highly dynamic interactions have also been to the study of the nucleus. While whole genome information is
characterized for the structural linker histone H1 and the HMG readily available, proteomic methods are now widely applied to

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studies of the nucleus and have already revealed valuable Syndrome.(99) The two puzzling questions regarding lamino-
insight into the complex and dynamic protein composition of pathies are: how does a mutation in a structural component of
the nucleolus, splicing factor compartments and the nuclear the nucleus give rise to disease and how does a defect in a
envelope.(93–95) These lists of nuclear components now ubiquitous protein lead to highly tissue-specific diseases? One
provide the basis to build extensive computer models to common feature of laminopathies is that the most-affected
address key questions such as how the proteinaceous tissues are generally exposed to mechanical stress, i.e.
compartments of the nucleus or the non-random spatial muscle, adipose tissues and skin, and this observation has
organization of genomes are established and maintained and lead to the proposal that laminopathies cause disease by
it will be important to ask whether nuclear organization can be weakening the mechanical stability of nuclei, thus leading to
reproduced in silico assuming the constraints of self-organiz- cellular defects and possibly cell death or alterations in gene
ing systems. Regardless of whether these bioinformatic and expression patterns (Fig. 3). In support, nuclei from LMNA/
computational approaches will unambiguously identify the mice indeed are more susceptible to mechanical stress.(100)
nucleus, and potentially the cell, as a self-organizing system, An alternative, but not mutually exclusive, possibility is that
their application will contribute greatly to uncover the basic disease-related defects in the lamina directly cause mis-
principles that govern cellular organization and function. regulation of genes (Fig. 3). This could occur by defects in
tethering of genes and their subsequent mis-expression or via
The concept of nuclear diseases a function of lamins in transcription and replication.(80,99)
Regardless of whether maintenance of nuclear architecture is
the result of rigid structural scaffolds or occurs largely by self-
reinforcing mechanisms, a key development in the recent
study of nuclear architecture has been the realization that
nuclear architecture contributes to human disease and that, in
some cases, defects in nuclear architecture directly cause
disease.
Numerous disease-related proteins localize to the nucleus
and their distribution is often altered in the disease state.(96)
Most prominently, in promyelocytic leukemia, the PML protein,
which normally localizes in 10–20 prominent nuclear PML
bodies, becomes dispersed through the nucleus. PML is also
mislocalized and mis-expressed in several solid tumors,
suggesting a more general role in tumors.(97) Despite the
clear correlation between PML localization and disease, the
role of PML in tumor formation is unclear. Another type of
nuclear disease are the poly(A)- and poly(Q)-repeat diseases
including X-linked mental retardation and Huntington’s dis-
eases.(98) These diseases are characterized by the presence
of proteins with expanded polyalanine or polyglutamine tracts.
These proteins frequently accumulate in the cell nucleus
where they form insoluble inclusion bodies. Whether these
nuclear bodies are cause or consequence of the aberrant
function of these proteins is not known. Apart from providing a
starting point for the elucidation of disease mechanisms,
morphological alterations of the nucleus, and mislocalization
or differential abundance of nuclear proteins is now being
increasingly pursued for diagnostic purposes.
The most-striking class of nuclear diseases, however, are Figure 3. Pathways to disease in human laminopathies.
the laminopathies.(99) This diverse class of diseases is caused Mutations in lamin A may lead to disease by causing
by mutations in the structural lamin A and C proteins encoded mechanical defects in the nuclear lamina, which result in cell
death or alterations in gene expression. Alternatively, mutations
by the LMNA gene and several lamin-associated proteins
in lamin A may also directly lead to aberrant gene expression,
including emerin and LAP2 (lamin-associated protein 2). possibly due to defects in the ability of genes to be properly
Mutations in these genes give rise to muscular dystro- tethered to the lamina network or to defects in transcription,
phies, lipodystrophies, neuropathies and, most intriguingly, replication and DNA repair.
the premature-aging disease Hutchinson-Gilford Progeria

484 BioEssays 27.5

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Detailed studies on mis-regulated gene in laminopathies will 14. Cremer T, Cremer C. 2001. Chromosome territories, nuclear architec-
hopefully distinguish between these possibilities. ture and gene regulation in mammalian cells. Nat Rev Genet 2:292–
301.
15. Parada L, Misteli T. 2002. Chromosome positioning in the interphase
Towards an integrated view of genome function nucleus. Trends Cell Biol 12:425.
As we learn more about the molecular mechanism of gene 16. Cremer T, Cremer C, Schneider T, Baumann H, Hens L, et al. 1982.
Analysis of chromosome positions in the interphase nucleus of Chinese
expression, the spatial organization of the genome and the hamster cells by laser-UV-microirradiation experiments. Hum Genet 62:
structure of the cell nucleus, we realize the tremendous 201–209.
complexity of the cell nucleus. However, several dominant 17. Manuelidis L. 1985. Individual interphase chromosome domains
revealed by in-situ hybridization. Hum Genet 71:288–293.
concepts that govern much of nuclear structure and function 18. van Driel R, Fransz PF, Verschure PJ. 2003. The eukaryotic genome:
have emerged and these will serve as starting points and a system regulated at different hierarchical levels. J Cell Sci 116:4067–
guideposts as we further explore how genomes function in 4075.
19. Kozubek S, Lukasova E, Jirsova P, Koutna I, Kozubek M, et al. 2002. 3D
vivo. The confluence of molecular studies, genome sequence Structure of the human genome: order in randomness. Chromosoma
information and cell biological insights is leading us rapidly 111:321–331.
towards an integrated view of genome function in vivo. This 20. Boyle S, Gilchrist S, Bridger JM, Mahy NL, Ellis JA, et al. 2001. The
spatial organization of human chromosomes within the nuclei of normal
development and the ever-growing theoretical framework and emerin-mutant cells. Hum Mol Genet 10:211–219.
provided by the emerging concepts in nuclear architecture 21. Tanabe H, Muller S, Neusser M, von Hase J, Calcagno E, et al. 2002.
form the basis to expand the studies of nuclear architecture Evolutionary conservation of chromosome territory arrangements in
cell nuclei from higher primates. Proc Natl Acad Sci USA 99:4424–
from the analysis of tissue cultured systems to physiologically 4429.
more relevant paradigms, including differentiation and dis- 22. Sun HB, Shen J, Yokota H. 2000. Size-dependent positioning of human
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23. Parada L, McQueen P, Munson P, Misteli T. 2002. Conservation of
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I thank members of my laboratory for their typically critical Inheritance of gene density-related higher order chromatin arrange-
comments on this manuscript and for continuously questioning ments in normal and tumor cell nuclei. J Cell Biol 162:809–820.
26. Roix JJ, McQueen PG, Munson PJ, Parada LA, Misteli T. 2003. Spatial
established concepts. TM is a Fellow of the Keith R. Porter proximity of translocation-prone gene loci in human lymphomas. Nat
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