Concepts in nuclear architectureTom Misteli

SummaryGenomes are defined by their primary sequence. Thefunctional properties of genomes, however, are deter-mined by far more complex mechanisms and depend onmultiple layers of regulatory control processes. A keyemerging contributor to genome function is the architec-tural organization of the cell nucleus. The spatial andtemporal behavior of genomes and their regulatoryproteins are now being recognized as important, yet stillpoorly understood, control mechanisms in genomefunction. Combined cell biological, molecular and com-putational analysis of architectural aspects of genomefunction has added a further dimension to the investiga-tion of some of the most fundamental cellular processesincluding transcription and maintenance of genomeintegrity. The complete elucidation of the contributionthat nuclear architecture makes to gene expression willbe required to fully understand physiological processessuch as differentiation, development and disease at thecellular level. Here I give an overview of some of theemerging concepts in the study of in vivo genomeorganization and function. BioEssays 27:477–487,2005. Published 2005 Wiley Periodicals, Inc.{

Introduction

Genomes function in the context of nuclear architecture.

Recent work has lead to the realization that the mammalian

cell nucleus is a spatially and functionally compartmentalized

organelle containing numerous proteinaceous nuclear bodies

as well as non-randomly positioned genome domains. The

spatial and temporal aspects of genome organization have

likely functional consequences as indicated by the fact that

changes in nuclear architecture are amongst the most dra-

matic hallmarks of development and differentiation processes

and defects in architectural elements of the cell nucleus are

now known to be responsible for several human diseases.

The key question of how genome function is integrated into

the architectural framework of the cell nucleus and how

structural elements of the nucleus affect nuclear processes

including gene expression, DNA repair and genome stability

are critical to our understanding of genome function. The

intense study of nuclear architecture has lead to the

emergence of several general concepts, many of them

surprising and provocative. I give here a brief overview of the

most central ones. I deliberately do not discuss any one

concept in detail, but aim to provide a broad overview of the

general ideas that form the framework for our understanding of

nuclear function. For a detailed discourse of each topic, I refer

the reader to several excellent recent review articles.

The concept of nuclear compartments

A central feature of the mammalian cell nucleus is its

morphological and functional heterogeneity generated by the

presence of distinct nuclear compartments.(1,2) A nuclear

compartment is defined as a macroscopic region within the

nucleus that is morphologically and/or functionally distinct

from its surrounding. Compartments are generally proteinac-

eous nuclear bodies or chromatin domains (Fig. 1).

Proteinaceous nuclear ‘bodies’Nuclear compartments are membraneless suborganelles

characterized by a distinct set of resident proteins. The most

prominent nuclear bodies include the nucleolus, which is the

site of transcription and processing of ribosomal RNA, the

splicing factor compartments, which act as a storage/

assembly site for spliceosomal components, the Cajal body,

which is the proposed site of snRNP assembly and the PML

body, which is of unknown function.(1) In addition to these

structures, many additional nuclear bodies characterized by

the presence of one or multiple nuclear proteins have been

described in the literature.(2) The function of many of these

bodies remains elusive. However, conceptually at least three

models must be considered for the function of a nuclear body.

First, the body may not be of functional relevance. The obser-

ved morphological bodies may simply be a consequence of

aggregation of excess protein that is not used (Fig. 1). The

observation that overexpression of some nuclear proteins can

result in the formation of structures that appear by fluores-

cence microscopy indistinguishable from nuclear bodies and

the fact that in some cases overexpression of nuclear proteins

results in the de novo formation of nuclear bodies are

consistent with this notion.(3) Second, a nuclear body may be

a site of particular nuclear activities. Thismodel clearly applies

to the nucleolus, which contains all ribosomal genes and is 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

suggested that they are sites of assembly of transcription

complexes.(5–7) A thirdmodel suggests that anuclear body is a

site of inactivity and may serve as storage site to provide

components to its immediate surroundings. This model has

National Cancer Institute, NIH, Bethesda, MD 20892.

E-mail: mistelit@mail.nih.gov

DOI 10.1002/bies.20226

Published online in Wiley InterScience (www.interscience.wiley.com).

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

been applied to the splicing factor compartments.(8,9) These

compartments are enriched in pre-mRNA splicing factors but,

enigmatically, were found not to be sites of pre-mRNAsplicing.

However, upon activation of genes in the vicinity of these

compartments, splicing factors are recruited from the com-

partments to the activated sites of transcription, suggesting

that the compartment serves as a reservoir of factors without

being an active site of splicing.(10) In this function, compart-

ments might contribute to regulate the concentration of

available factors in the nucleoplasm and at their actual sites

of action.(8)

The absence of membranes provokes the question of how

nuclear compartments are formed and maintained. One

possibility is that the compartments contain structural proteins

that serve as a stable scaffold around which a nuclear body is

formed. However, no skeletal proteins have been identified for

any of the known nuclear bodies. An alternative possibility is

that nuclear bodies are largely the results of the sum of many,

likely transient and non-specific, interactions amongst its

resident proteins. In support of such a self-organizing model,

the potential for self-interaction has been reported for marker

proteins of virtually all nuclear bodies.(1,11)

Chromatin domainsIn addition to proteinaceous bodies, large-scale chromatin

domains exist within the nucleus. The major chromatin

domains are morphologically defined as euchromatin consist-

ing of less-condensed genome regions, whereas heterochro-

matin contains more highly condensed regions (Fig. 1).

Euchromatin is generally considered to be the site of ‘open’,

presumably transcriptionally active, chromatin regions and

heterochromatin is often thought of enriched in inactive and

silenced genome regions. While the two types of chromatin

can easily be defined based on their morphology, it is

becoming increasingly clear that the correlation of large-scale

chromatin morphology and function is an oversimplification. A

recent study by Bickmore and colleagues has directly compar-

ed the relationship of chromatin structure with gene activity on

a genome-wide scale.(12) Using biochemically defined probes,

the distribution of ‘open’, ‘closed’ and ‘bulk’ chromatin were

mapped on intact chromosomes and onto DNA chips. Rather

than finding a strong correlation between chromatin structure

and gene activity, a link between chromatin morphology and

gene density was found with gene-rich regions being present

in open chromatin regions and gene-poor regions in more-

condensed domains, regardless of their activity status. It thus

appears that the view of euchromatin as regions of genome

activity and heterochromatin as regions of genome inactivity is

oversimplistic.(12)

Heterochromatin and euchromatin can also, roughly, be

defined molecularly based on differential post-translational

modifications of core histones.(13) Euchromatin is generally

enriched in hyperacetylated histones H3 and H4, whereas

heterochromatin is characterized by hypoacetylation and tri-

methylation of histoneH3 on lysine 9. The latter modification is

thought to serve as a mark for heterochromatin and as a

binding site for structural heterochromatin proteins, which

likely contribute to maintaining the highly condensed state of

chromatin. How precisely the morphological, biochemical and

molecular definitions of chromatin domains correspond to the

functional status of the various chromatin regions is an issue of

current investigation.

The concept of chromosome territories

Chromosomes exist in the nucleus in the form of chromosome

territories.(14,15) This definition denotes the fact that the

genetic material of each chromosome is not widely distributed

throughout the nucleus but occupies a spatially defined

subvolume (Fig. 1). The existence of chromosome territories

was originally demonstrated by inducing DNA damage in a

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 caneither benon-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).

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478 BioEssays 27.5

defined region of the nucleus using a microlaser and analysis

of the damaged chromosomes in the subsequent meta-

phase.(16) These ingenious experiments revealed that only a

small subset of chromosomes was damaged and that the

number of chromosomes was dependent on the size of the

irradiated area. These findings were most consistent with a

model in which chromosomes are arranged in spatially limited,

well-defined, nuclear subvolumes. This conclusion was later

directly confirmed by fluorescence in situ hybridization using

probes specific for entire chromosomes, which allowed the

visualization of chromosome territories in situ.(17)

Despite the fact that the outlines of the chromosome

territories are spatially well defined their internal structure is

still ambiguous.(18) Initially thought of as solid domainswith the

active transcribed genes at the surface and exposed to

regulatory factors thatmight be present in canal-like structures

between chromosome territories, it is now clear that chromo-

some territories are not solid structures, but rather are

permeated by nucleoplasmic channels of various sizes.(14)

This creates a porous entity with a highly convoluted and

enlarged surface area. This structural property likely facilitates

access of regulatory factors to sequences buried within the

chromosome territory.Chromosome territories are not uniform

in their structure and appearance and likely contain loops of

varying sizes leading to intermingling between neighboring

territories.(18) As a corollary of the discrete nature of chromo-

some territories, the possibility arises that chromosomes are

arranged in particular patterns within the nuclear space.(15)

The concept of non-random nuclear organization

One of the major emerging concepts in the study of nuclear

architecture is the non-random organization of the nuclear

space. Themammalian cell nucleus is highly non-randomboth

in its arrangement of the genome, of nuclear compartments

and of nuclear proteins. The non-random nature of nuclear

organization is oneof themost striking andpuzzling featuresof

the mammalian cell nucleus and the functional significance of

non-randomnuclear organization is oneof the current frontiers

in the field.

Genomes in three dimensionsGenomes are non-randomly arranged in three-dimensional

space.(14,15,19) A convenient, although highly simplistic,

indicator of the position of a chromosome or a locus is the

radial position, i.e. its location relative to the nuclear center or

the periphery (Fig. 2A). Amongst many other examples, in

lymphocytes and fibroblasts, the radial position has been

correlated with a chromosome’s gene density, with gene-rich

chromosomes positioned preferentially towards the center of

the nucleus, and gene-poor chromosomes located towards

the periphery.(19–21) In other studies in fibroblasts, radial gene

positioning has been correlated with chromosome size rather

than gene density.(22) The non-random arrangement of

chromosomes is also reflected in the positioning of chromo-

somes relative to each other (Fig. 2A). In mouse lymphocytes,

for example, single homologues of chromosomes 12, 14 and

15 frequently form a non-random cluster.(23) This cluster only

contains one of the two homologueswith the others apparently

randomly distributed throughout the nuclear volume. Interest-

ingly, tetraploid cells frequently contain two such clusters,

suggesting that distinct rules of positioning apply to chromo-

some homologues.(23)

The arrangement of chromosomes within the nucleus

appears to be tissue-specific.(20,24,25) A systematic study of

multiple chromosomes in several primary tissues has shown

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 regionmayeither be a silencing environment such as

heterochromatin or an active environment such as a transcription factory. Associationwith the particular enviromentmay results in changes

in the functional status of the gene (red).

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BioEssays 27.5 479

chromosomes relative to each other differs amongst tis-

sues.(24) Statistical comparison of distribution patterns further

suggests that cell types, which share differentiation pathways,

such as lymphocytes and myeloblasts, exhibit a more similar

three-dimensional organization of genomes than unrelated

cell types.(24) While these observations suggest a role for

differentiation in determining positioning patterns, the exact

mechanisms that give rise to non-random chromosome

positioning are unknown.

An underlying concept in understanding three-dimensional

genome organization is the probabilistic nature of positioning

patterns. The radial or relative position of a chromosome is

always a reflection of its preferred position, but does not

indicate that a given chromosome is found in one particular

place in all cells of a population. Thus, while it should be

possible to generate probability maps of how genomes are

spatially organized, it will not be possible to define the position

of a genome region in absolute terms.

Gene positioningSine chromosomes are non-randomly arranged in the

nucleus, it is not surprising that the gene loci that they harbor

are also found in non-random patterns. How the position of a

locus relative to the nuclear periphery or relative to nuclear

landmarks, such as chromatin domains or the chromosome

territory, affects its function is one of the key questions in

understanding the contribution of spatial positioning to

genome function.

Although strong preferential positioning relative to the

nuclear center has been observed for many analyzed genes,

this positioning is likely not critical for function, since a

particular gene, regardless of whether it is active or inactive,

can be found at virtually any position in the nucleuswithin a cell

population.(19,26–28) It seems more likely that the non-random

positioning observed for loci is largely the consequence of

the positioning of the chromosomes on which the gene is

found. On the other hand, changes in the functional status of

several genes have been reported to be paralleled by

repositioning. For example, in T-cell differentiation, the CD4

locus moves towards the periphery as it becomes inactivated

during selection for CD8þ cells.(27) However, repositioning is

clearly not a pre-requisite for changes in function since, in the

same cells, the CD8 locus does not alter its intermediate radial

position regardless of its functional status.(27)

More important than radial positioningmight be tethering of

gene loci to nuclear landmarks (Fig. 2B). In yeast, association

of gene loci with the nuclear periphery occurs in response to

both gene activation and gene inactivation depending on the

gene.(29) Furthermore, in yeast and Drosophila, boundary

elements that functionally insulate neighboring genome

regions appear to act by tethering the affected regions to a

nuclear structure such as the nuclear periphery or the

nucleolus(30,31) (Fig. 2B).

Possibly the functionally most important type of positioning

is the location of a gene relative to chromatin domains,

particularly transcriptionally repressed heterochromatin do-

mains. The classic example of positioning-induced silencing is

thephenomenonof positioneffect variegation (PEV). InPEV, a

locus becomes permanently silenced due to physical place-

ment into a heterochromatic genome neighborhood. In the

case of the well-studied brown locus inDrosophila insertion of

a heterochromatin block near the locus results in its physical

positioning near a heterochromatin region and its silencing

in transvia contactwith theexisting heterochromatin domain in

a sequence-independent manner.(32) Thus, positioning rela-

tive to heterochromatin domain can clearly have a regulatory

effect. This effect is most likely not limited to artificial experi-

mental systems, since numerous examples of correlations

between proximal positioning of a locus and its silencing have

been reported in differentiation systems.(33) Having said that,

association with a heterochromatin region does not necessa-

rily confer transcriptional silencing, since some loci, such as

the l-5 locus in B-cells, retain their activity despite positioning

within heterochromatin regions and a significant fraction of

genes are found in heterochromatin in Drosophila.(34)

It is not surprising to find that gene loci generally localize

within the chromosome territory on which they localize.

However, this is not always the case and both the dissocia-

tion from territories as well as the preferential positioning of

genome regions at the surface of territories has been sug-

gested to contribute to proper gene function(35,36) (Fig. 2C).

In human and mouse cells, several genome regions have

been characterized that are expelled from their chromosome

territory and loop several micrometers away from the main

body of the chromosome.(37–39) All of these domains contain

gene clusters and are highly transcribed. Looping likely

contributes to gene regulation as suggested by analysis of

the correlation between loop formation and gene activity

during differentiation processes. In erythroid cells, theb-globinlocus loops out from the chromosome territory before it is

transcriptionally active, but remains associated with the

chromosome when its regulatory elements are mutated.(35,40)

Similarly, in differentiating ES-cells, theHox1 andHox 9 genes

dissociate from their territories in parallel with their activity.(41)

While the former example suggests looping as a consequence

of potentiation of the gene, the latter points towards a correla-

tion with gene activity per se (Fig. 2C). Whether these exam-

ples represent different paradigms of positioning and how

common thedifferent behaviorsare remains to beestablished.

Genome regions may also be non-randomly positioned

relative to each other. The best example of spatial clustering of

genes is the ribosomal genes, which congregate non-

randomly in the nucleolus. A typical mammalian nucleolus

contains 50–100 active ribosomal genes located on 2–4

distinct chromosomes.(4) While it seems clear that the

clustering of ribosomal genes enhances the efficiency of

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480 BioEssays 27.5

rRNA transcription and processing, it is not known what the

mechanisms are that lead to the clustering of the chromo-

somes. Similar clustering has recently been observed for

tRNAgene inS. cerevisiae, wheremore than50genes located

on all 16 chromosomes cluster near the nucleolus.(42) Initial

evidence for spatial clustering of RNA pol II genes in

mammalian cells has recently also been reported.(40) Osborne

et al., have found that two genes located 25 Mb apart on

chromosome 7 pair in three-dimensional space to generally

share a common transcription domain. This observation

suggests that spatial gene clusters might be more prevalent

than previously expected.

The positioning of genome regions, be it chromosomes,

genome regions or single genes,might not only be functionally

relevant for gene expression, but especially for cellular

processes that involve the physical interaction of genome

regions such as formation of translocations or recombina-

tion.(43) In support of an important role of non-random

positioning is the fact that strong correlations have been

detected between the spatial proximity of chromosomes or

genes and their probability of undergoing translocations or

recombination. In human cells, the translocation partners for

several lymphomashavebeen found in closer spatial proximity

than would be expected based on their random distribu-

tion.(26–46) Similarly, in yeast, the preferred recombination

partners involved in preferential donor selection during

mating-type switching are on average in closer spatial

proximity than unrelated loci.(47) These observations suggest

that the non-random spatial positioning of genome regions in

the interphase nucleus may contribute to determining what

genome region interact with what other regions.(15)

Taken together, the role of positioning in gene function is still

unclear. A major impediment towards deducing general rules

for how positioning affects gene function has been the

limitation of most studies to the analysis of single genes

resulting in largely anecdotal reports of correlation between

positioning and function. More systematic analysis of multiple,

functionally related genes using high-throughput imaging

methods and pattern recognition tools will likely resolve this

issue in the future. In addition, the mining of whole genome

sequence information to understand how the primary genome

sequence affects higher order organization of chromatin and

its environment and to ask whether higher order structure can

be predicted from sequence information will be a necessary

and promising, albeit challenging, approach to understand

spatial genome organization and its link to function.

Positioning of nuclear bodiesNot only are genomes non-random arranged in vivo, but

increasing evidence also suggests that non-random position-

ing of nuclear bodies relative to specific genome regions is the

norm rather than the exception. The clearest example of non-

random positioning of a nuclear body to gene loci is the

nucleolus, where ribosomal genes are transcribed and the

resulting rRNAs processed.(4) The nucleolus invariably as-

sociates with the subset of chromosomes containing the

tandemly repeated rDNA clusters. The association is driven by

the transcriptional activity of the ribosomal genes since

introduction of rDNA genes on non-integrating plasmids leads

to the formation of mini-nucleoli and inhibition of the ribosomal

DNA-specific RNA polymerase I results in disassembly of

nucleoli.(4) Furthermore, the nucleolus disassembles during

mitosis when rDNA genes are silenced, but rapidly reforms

upon re-imitation of ribosomal gene activity in late telophase.

Analogous to the situation of the nucleolus, Cajal bodies

appear to associate with relatively high frequency with histone

and U2 snRNA gene clusters and introduction of U2 snRNA

minigenes promotes the association of Cajal Bodies.(48–50)

These observations on nucleoli and Cajal bodies strongly

suggest that the non-random association of these nuclear

compartments is a direct consequence of the transcriptional

status of the target genes.

Other nuclear bodies have been found in non-random

proximity with genome regions, but it is not clear whether this

association is driven by specific genes or by broader genome

regions. For example, the OPT-domain (Oct1/ PTB/transcrip-

tion) has been mapped to preferentially associate with a �30

Mb region on human chromosome 6p21.(51) A particularly

intriguing association is that of the heat-shockgranules. These

structures form in human cells upon heat shock. They

preferentially form at a satellite repeat region on chromosome

9 and, remarkably, concomitant with heat shock this normally

silent region becomes transcriptionally active and generates

an apparently non-coding RNA of unknown function.(52,53) In

this case, it is not clear whether formation of the heat-shock

granule drives transcription from this site or whether it

represents the consequence of transcriptional activation of

the satellite region.

Several nuclear structures associate non-randomly,

although more generally, with active genome regions. PML

bodies are preferentially found near active genes and splicing

factor compartments containing pre-mRNA splicing factors

often associate with gene-dense R-bands.(54,55) While the

association of PML bodies with active genes does not appear

to be functionally important since depletion of PML has no

affect on theassociation, it seemshighly likely that thephysical

proximity of splicing factor compartments facilitates the

processing of transcription generated from the gene-dense

R-bands. It thus appears that the non-random association of a

nuclear body with a genome region is generally the conse-

quence of the region’s activity.

The concept of nuclear dynamics

The cell nucleus has recently emerged as a highly dynamic

organelle and it is now clear that virtually all aspects of nuclear

function and organization are dynamic.(56) Photobleaching

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BioEssays 27.5 481

experiments have revealed that proteins roam the nuclear

space in an energy-independent manner in search for high-

affinity binding sites.(57) This ability of molecules to freely

diffuse through the nuclear space appears particularly

important for chromatin-binding proteins.(58–61) These factors

have been shown to undergo highly transient binding interac-

tions with chromatin both at their specific as well as their non-

specific binding sites. Estimated residence times for proteins

on chromatin, including structural proteins of euchromatin and

heterochromatin, are typically on the order of 2–30 s and the

time between binding events is on the order of 50–200 ms.(58)

The latter value indicates that, once dissociated fromabinding

site, a chromatin protein is rapidly capturedbyanon-specific or

a specific binding site in its vicinity. An implication of this

behavior is that at any given time a large fraction of any

chromatin protein is bound, most frequently to a non-specific

site, and that only a small fraction of the protein is unbound.(58)

The combined transient interaction and theability of proteins to

diffuse rapidly suggest that chromatin proteins scan the

genome for appropriate high-affinity binding sites by a three-

dimensional hopping mechanism.(58)

The molecular basis for the dynamic nature of protein–

chromatin interactions is unclear. Evidence from the analysis

of dynamic binding of steroid receptors in permeabilized cells

suggests that the rapid turnover of steroid receptors is

promoted by chaperone activities that either directly act on

the receptors to remove them or indirectly cause their

dissociation by acting on some of their interaction partners.(62)

In addition, evidence from in vitro reconstitution and ultra-fast

crosslinking experiments has also suggested that the resi-

dence time of chromatin proteins may be related to chromatin

remodeling, which might periodically destabilize the weak

binding of chromatin proteins.(63) In vivo observations using

inhibitors have pointed towards a direct or indirect role for

proteasome activity in the dynamic exchange of chromatin

proteins.(60,64) Given the large variety of chromatin-binding

proteins, it seems likely that the mechanisms of exchange will

greatly vary between different proteins. Regardless of the

detailed molecular mechanisms for the transient protein–

chromatin interactions, the functional significance of transient

binding is likely the ability of these proteins to undergomultiple,

rapidly changing interactions with partners on chromatin to

generate combinatorial protein complexes on chromatin.(56,58)

This property likely allows cells to alter their transcriptional

output rapidly in response to stimuli and thus provides a

molecular basis for the high transcriptional plasticity inherent

to most cellular systems.

Photobleaching experiments have also uncovered that

proteinaceous nuclear bodies including the nucleolus are

generated from rapidly exchanging molecular compo-

nents.(57,65,66) Similar to the situation for chromatin proteins,

it seems now most likely that ‘resident’ nuclear body proteins,

diffuse relatively freely throughout the nucleoplasm until they

interact with their ‘home’ compartment where they are

captured by higher-affinity interactions than they encounter

in the rest of the nucleus.(56) This interpretation is consistent

with the discovery that many resident proteins of nuclear

bodies frequently visit other nuclear structures.(67) Thus, the

overall stable nuclear suborganelles are generated from

dynamic components.

Not only proteins and protein complexes exhibit dynamic

motion in vivo, but nuclear bodies themselves are dynamic.

Although the nucleolus is positionally very stable and does not

change its location over short periods of time, most other

nuclear bodies do. Cajal bodies undergo saltatory motion and

PML bodies have been reported to be able to move in linear

trajectories through the nucleus.(68,72) Both of these move-

ments are sensitive to ATP levels, but it is difficult to ascertain

that this effect reflects an energy dependence of the motion

itself or is an indirect effect caused by the perturbation of large-

scale chromatin structure due to ATP depletion.(69,71,72) What

the functional significance of nuclear bodymovementmight be

is unclear, but it has been suggested that, similar to the

situation for chromatin-binding proteins, the movement of

nuclear bodiesallows them to scan thegenome for preferential

interaction sites.(69) This idea is consistent with the observed

non-random association of nuclear bodies with particular

genome regions.

Gene loci themselves can undergo dynamicmotion aswell.

In vivo tracking of fluorescently tagged loci demonstrates that

they undergo significant constraint diffusionalmotion.(73–75) In

mammalian cells, a locus may explore its immediate environ-

ment by local motion within a subvolume of typically about

1–2 mm in diameter.(75) Remarkably, the absolute range of

motion of a given locus is similar in mammalian cells and

yeast.(74) A locus can deviate from its average position by

about 1 mm over the period of several minutes. However, in

absolute terms, this motion has very distinct significance in

mammals and in yeast, since in mammals this motion

corresponds to sampling of a volume roughly equivalent to a

single chromosome, whereas in yeast this represents an

exploration of the entire cell nucleus. The motion of a locus

may be reduced by tethering to the nuclear periphery or

intranuclear structures such as nucleoli.(75) This is particularly

evident by the immobilization at the nuclear envelope of

telomeres in yeast.(74) The measured diffusion coefficients of

themotion of a locus are orders of magnitude lower than those

of proteins and thus a locus largely appears immobile relative

to diffusing proteins. Whether the diffusional mobility of

genome loci has any functional significance is unclear.

The concept of nuclear scaffolds

The complex spatial organization of the nucleus has given rise

to the tempting concept of a structural framework in the form of

a karyoskeleton or matrix that might contribute to spatially

organize the multitude of nuclear compartments.(76,77) The

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482 BioEssays 27.5

molecular identify of sucha structural scaffold remains elusive.

Several interesting candidates have been identified. In

Drosophila, the non-chromosomal EAST protein has been

suggested to be part, or at least control, a putative nuclear

endoskeleton, since overexpression of the protein causes

expansion of the extrachromosomal space and the nuclear

volume.(78) In human thymocytes, the SATB1 protein appears

to formacage-likenetworkof proteinaceous filaments towhich

genes can be attached.(79) The association of several

thymocyte-specific genes appears to correlate with their

activity suggesting that association with this scaffold has a

regulatory function.

Anobvious candidate for a scaffold component are thewell-

characterized intermediate-filament-type lamin proteins,

which have long been known to form a structural meshwork

underlying the nuclear envelope refereed to as the nuclear

lamina.(80) Recent observations strongly suggest that lamins

A,BandCarealsopresent in the interior of thenucleusandare

functionally critical for transcription and replication.(80) It is

unclear what their contribution to the organization of nuclear

structures is at present, but at least lamins A and C are not

essential for the formationof splicing factor compartments and

other nuclear bodies.(81,82) A further prominent candidate for a

structural component of a karyoskeleton is actin. It is now

established that monomeric actin is present in the nucleus and

appears to be functionally relevant in transcription complex

formation; whether actin filaments exist in the nucleus is not

known.(83)

The concept of nuclear self-organization

The difficulties in unambiguously defining a static nuclear

skeleton and the observed highly dynamic nature of nuclear

architecture has lead to the consideration of suggestions that

the nucleus may be a self-organizing entity.(84,85) In this

model, the complex morphological appearance is a reflection

of the transcriptional activity of the genome and the sum

of all molecular interactions amongst nuclear components.

Although this provocative model is speculative at present,

virtually all observations on nuclear organization and

dynamics are consistent with it. Central to self-organizing

systems is the high dynamic content and a relative promiscuity

of interactions amongst components. The recent observations

of the dynamics of numerous nuclear proteins in living cells

clearly supports both of these premises. As discussed above,

proteins can rapidly roam the nuclear space, the resident

components of subcompartments are dynamically exchanged

and many chromatin proteins undergo extensive non-specific

transient interactions with chromatin and with nuclear

bodies.(56) Consistent with a self-organization model, the

major proteins that confer the structural identity of hetero-

chromatin exchange and stochastically find their specific

binding site.(86,87) Highly dynamic interactions have also been

characterized for the structural linker histone H1 and the HMG

proteins, which compete with each other for binding sites in a

dynamic interaction network.(88) These dynamic, largely

random interactions amongst proteins are consistent with

the recent realization that gene expression events are highly

stochastic.(89,90)

The principle of self-organization is also consistent with the

observed probabilistic nature of chromosome positioning and

can also account for the non-random positioning of genes

relative to nuclear landmarks. In this view, positioning may be

the result of themutual interplay between the functional status

of a locus and the nuclear domain with which it interacts

(Fig. 2C). A plausible scenario is that a gene locus becomes

potentiated by specific local events such as binding of par-

ticular regulators or histone modifications. The potentiated

locus may then be incorporated into a more ‘open‘ chromatin

loop, which, in the extreme case, may become expelled from

the chromosome territory. The more flexible loop is now free

toassociatewith functionally distinct subregionsof thenucleus

and may become preferentially associated with domains that

are of functional equivalence. The interaction of the locus with

these domains then in turn may stabilize its functional status,

resulting in a non-random preferential positioning.(43)

The auto-reinforcing behavior of self-organizing systems

may contribute greatly to the overall stability of nuclear

structure and the functional status of the genome but, at the

same time, the transient nature of virtually all protein–protein

and protein–chromatin interactionsmayalso poise the system

for rapid change in response to external stimuli. Thus, the

dynamic, self-organized, nature of nuclear organization is a

fundamental, functionally essential property of the cell.

Self-organization models are notoriously difficult to experi-

mentally test or falsify. While it is hard to imagine a single key

experiment to prove that the nucleus is a self-organizing

system, combining information from experimental and com-

putational approaches will likely allow critical probing of this

proposal. Biochemical experiments will tell us whether a

nucleus is able to self-organize into a functional entity from its

components as would be predicted. Simple nuclear re-

assembly systems capable of transport across themembrane

and transcription of defined DNA have been used for years.(91)

Furthermore, it has been demonstrated that nucleoli and PML

bodies dispersed by incubation in a low monovalent cation

environment can reassemble and resume their cellular

functionality by simple addition of inert macromolecules,

suggesting that both the structural and functional integrity of

nuclear compartments depends strongly on molecular crowd-

ing events as would be expected for a self-organizing

system.(92) In addition to direct experimental interrogation,

the concept of self-organization will also be tested by the

converging efforts of system biology approaches and compu-

tationalmodelingmethods,whicharenow increasinglyapplied

to the study of the nucleus.While whole genome information is

readily available, proteomicmethods are nowwidely applied to

Review articles

BioEssays 27.5 483

studies of the nucleus and have already revealed valuable

insight into the complex and dynamic protein composition of

the nucleolus, splicing factor compartments and the nuclear

envelope.(93–95) These lists of nuclear components now

provide the basis to build extensive computer models to

address key questions such as how the proteinaceous

compartments of the nucleus or the non-random spatial

organization of genomes are established and maintained and

it will be important to ask whether nuclear organization can be

reproduced in silico assuming the constraints of self-organiz-

ing systems. Regardless of whether these bioinformatic and

computational approaches will unambiguously identify the

nucleus, and potentially the cell, as a self-organizing system,

their application will contribute greatly to uncover the basic

principles that govern cellular organization and function.

The concept of nuclear diseases

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

the laminopathies.(99) This diverse class of diseases is caused

by mutations in the structural lamin A and C proteins encoded

by the LMNA gene and several lamin-associated proteins

including emerin and LAP2 (lamin-associated protein 2).

Mutations in these genes give rise to muscular dystro-

phies, lipodystrophies, neuropathies and, most intriguingly,

the premature-aging disease Hutchinson-Gilford Progeria

Syndrome.(99) The two puzzling questions regarding lamino-

pathies are: how does a mutation in a structural component of

the nucleus give rise to disease and how does a defect in a

ubiquitous protein lead to highly tissue-specific diseases?One

common feature of laminopathies is that the most-affected

tissues are generally exposed to mechanical stress, i.e.

muscle, adipose tissues and skin, and this observation has

lead to the proposal that laminopathies cause disease by

weakening the mechanical stability of nuclei, thus leading to

cellular defects and possibly cell death or alterations in gene

expression patterns (Fig. 3). In support, nuclei from LMNA�/�

mice indeed are more susceptible to mechanical stress.(100)

An alternative, but not mutually exclusive, possibility is that

disease-related defects in the lamina directly cause mis-

regulation of genes (Fig. 3). This could occur by defects in

tethering of genes and their subsequent mis-expression or via

a function of lamins in transcription and replication.(80,99)

Figure 3. Pathways to disease in human laminopathies.

Mutations in lamin A may lead to disease by causing

mechanical defects in the nuclear lamina, which result in cell

deathor alterations in geneexpression.Alternatively,mutations

in lamin A may also directly lead to aberrant gene expression,

possibly due to defects in the ability of genes to be properly

tethered to the lamina network or to defects in transcription,

replication and DNA repair.

Review articles

484 BioEssays 27.5

Detailed studies on mis-regulated gene in laminopathies will

hopefully distinguish between these possibilities.

Towards an integrated view of genome function

As we learn more about the molecular mechanism of gene

expression, the spatial organization of the genome and the

structure of the cell nucleus, we realize the tremendous

complexity of the cell nucleus. However, several dominant

concepts that govern much of nuclear structure and function

have emerged and these will serve as starting points and

guideposts as we further explore how genomes function in

vivo. The confluence of molecular studies, genome sequence

information and cell biological insights is leading us rapidly

towards an integrated view of genome function in vivo. This

development and the ever-growing theoretical framework

provided by the emerging concepts in nuclear architecture

form the basis to expand the studies of nuclear architecture

from the analysis of tissue cultured systems to physiologically

more relevant paradigms, including differentiation and dis-

ease models. There is now little doubt that understanding

nuclear architecturewill be the cornerstone for the discoveryof

how genomes really work in living organisms.

Acknowledgments

I thank members of my laboratory for their typically critical

commentson thismanuscript and for continuously questioning

established concepts. TM is a Fellow of the Keith R. Porter

Endowment for Cell Biology.

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