Download The eukaryotic genome: a system regulated at different hierarchical

Commentary
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The eukaryotic genome: a system regulated at
different hierarchical levels
Roel van Driel*, Paul F. Fransz and Pernette J. Verschure
Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 318,1098SM Amsterdam, The Netherlands
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science 116, 4067-4075 © 2003 The Company of Biologists Ltd
doi:10.1242/jcs.00779
Summary
Eukaryotic gene expression can be viewed within a
conceptual framework in which regulatory mechanisms are
integrated at three hierarchical levels. The first is the
sequence level, i.e. the linear organization of transcription
units and regulatory sequences. Here, developmentally coregulated genes seem to be organized in clusters in the
genome, which constitute individual functional units. The
second is the chromatin level, which allows switching
between different functional states. Switching between a
state that suppresses transcription and one that is
permissive for gene activity probably occurs at the level of
the gene cluster, involving changes in chromatin structure
that are controlled by the interplay between histone
modification, DNA methylation, and a variety of repressive
and activating mechanisms. This regulatory level is
combined with control mechanisms that switch individual
genes in the cluster on and off, depending on the properties
of the promoter. The third level is the nuclear level, which
includes the dynamic 3D spatial organization of the genome
inside the cell nucleus. The nucleus is structurally and
functionally compartmentalized and epigenetic regulation
of gene expression may involve repositioning of loci in
the nucleus through changes in large-scale chromatin
structure.
Introduction
The genome sequences of an increasing number of organisms
are now known. Within the draft sequence of the human
genome (McPherson et al., 2001; Venter et al., 2001), most
protein-coding genes and a limited number of RNA genes have
been identified: together close to 35,000 genes. This number
will increase, because we probably underestimate the number
of genes that encode RNAs, of which many may be involved
in gene regulation (Marker et al., 2002). How the controlled
expression of the tens of thousands of genes in a genome is
orchestrated is a pressing question. This question is difficult to
answer owing to several fundamental problems, including the
following: (1) gene expression is controlled by regulatory
systems that act at different hierarchical levels, and we are only
beginning to appreciate how they are integrated; (2) gene
regulation involves precisely controlled changes in chromatin
structure, which are difficult to analyse in vivo; and (3) control
of gene expression in a particular cell depends not only on
DNA sequences but also on the history of that cell [e.g. during
embryonic development, epigenetic memory mechanisms
operate (Turner, 2002)]. A major challenge is to unravel the
language and syntax of the system that is responsible for
coordinated expression of so many genes. Here, we discuss
several aspects of hierarchical gene control in eukaryotes.
To explore the problem of how the genome functions it is
useful to discriminate three regulatory levels (Fig. 1). Level 1
is the sequence level, i.e. the 1D organization of functional
sequence elements in the genome. This level includes all
coding regions, the wide variety of regulatory sequences that
bind sequence-specific protein factors, and sequence elements
that may have a role in determining the 3D folding of the
chromatin fibre. Level 2 is the chromatin level and reflects the
different functional states chromatin can adopt in relation to
genome function. Classically, one might think about chromatin
structure in terms of euchromatin and heterochromatin.
However, it is likely that the chromatin fibre can exist in many
more different structural and functional states, which exhibit
different core histone compositions (Meneghini et al., 2003)
and post-translational modifications of histones (the ‘histone
code’) (Turner, 2002). Level 3 is the nuclear level, the 3D
structure and functional compartmentalization of the genome
inside the interphase nucleus. A classical example is the
nucleolus, in which the rRNA-coding gene clusters of several
chromosomes are brought together to create a subnuclear
domain that is dedicated to rRNA synthesis and processing and
pre-ribosome synthesis (Pederson and Politz, 2000). Another
example is the clustering of heterochromatin at particular
regions – for example, near the nuclear envelope.
Key words: Chromatin, Histone code, Nuclear organization, Gene
expression
The sequence level
The best-studied level of gene regulation is that of the
individual gene, involving cis-acting elements, such as
promoters, enhancers and silencers, in addition to trans-acting
factors, including DNA-binding transcription factors, cofactors, chromatin-remodelling systems and RNA polymerases
(Fig. 1). This complex machinery is tightly coupled to RNA
processing and RNA transport (Maniatis and Reed, 2002). A
fundamental question is whether the activity of genes is
controlled individually or whether genes are clustered and
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Journal of Cell Science 116 (20)
Fig. 1. Schematic representation of the three levels in genome
organization. (A) The sequence level. The black line is the DNA (fat
black lines represent coding regions, thin line intergenic DNA),
green bars are promoters and enhancers and blue boxes are boundary
elements. (B) The chromatin level. Red, yellow and white boxes
depict nucleosomes, blue boxes are boundary elements, green bars
are recruitment sites for proteins that change the histone code, or
otherwise induce a change in the structural and functional state of
chromatin. From here the change in chromatin state may spread
along the chromatin fiber, as indicated by the arrows. The red and the
yellow chromatin domains are in a different state. (C) The nuclear
level. The image shows part of a HeLa cell nucleus in which
chromatin was labelled by integration of GFP-histone H2B
(Verschure et al., 1999). Light areas (local high GFP-concentration)
represent condensed chromatin, whereas black areas correspond to
the interchromatin compartment. The red and the yellow chromatin
domains depicted in B have been drawn in the image. The yellow
domain is epigenetically silenced and is part of condensed
chromatin. The red domain is permissive for transcription and loops
out into the interchromatin space.
major regulatory decisions are made at the cluster level. A
limited number of gene clusters have been studied in detail –
for example, the α-globin genes, β-globin genes, histone genes
and Hox genes. Each cluster encodes a set of functionally
related proteins, and the genes are expressed in a coordinated
manner. Specific regulatory elements – for example, locus
control regions (LCRs), have been found to regulate gene
activity at the level of the cluster – in addition to those that
control expression of the individual genes (Festenstein and
Kioussis, 2000; Ho et al., 2002; Li et al., 2002). Although
clusters of functionally related genes are relatively rare,
growing evidence indicates that many genes are clustered not
according to their function but to when they are expressed –
for example, in a specific differentiation state.
Expression profiles in Drosophila support the notion of gene
clusters (Oliver et al., 2002; Spellman and Rubin, 2002). Under
a wide variety of different experimental conditions, groups of
on average 20 adjacent genes (representing domains of 20 to
200 kb) display very similar expression profiles, which
suggests that they are co-regulated. Remarkably, the genes in
such clusters have no obvious functional relationship, except
that they are expressed under the same conditions. About
20% of genes analysed are present in co-regulated clusters.
Analysing the expression of testes-specific genes in
Drosophila, Boutanaev et al. have drawn similar conclusions
about gene clustering (Boutanaev et al., 2002). Co-expressed
muscle-specific genes of C. elegans are also arranged in
clusters of two to five genes (Roy et al., 2002). These
observations may be just the tip of the iceberg, since it is likely
that regulation at the level of gene clusters primarily represents
switching of chromatin domains from a transcriptionally
repressive state (i.e. epigenetically silenced) to a state that is
permissive for transcription (see the section on the chromatin
level, below). Switching does not necessarily induce
transcriptional activation of the genes in the cluster; rather it
prepares them for regulation at the level of individual genes.
The expression profiling approaches that have been used in
identifying gene clusters (Boutanaev et al., 2002; Oliver et al.,
2002; Spellman and Rubin, 2002) pick up only clusters in
which all genes are transcriptionally activated (or inactivated)
simultaneously. Gene clusters that behave this way may be
relatively rare. These data nevertheless indicate that eukaryotic
genomes are functionally compartmentalized and that clusters
of genes can be co-activated and co-repressed.
How are gene clusters regulated? At least three classes of
genomic element should be involved in the control of cell-typespecific expression of gene clusters. First, a cluster-control
element that is responsible for switching the genomic domain
between its active and inactive state should be present. Such
regulatory elements may recruit histone-modifying enzymes,
causing a change in the functional state of chromatin in the
domain (see below). LCRs fulfil this role. These often-complex
sequence elements can switch genomic domains containing
one or more genes to a state that allows transcription to be
controlled by their respective promoters. LCRs have been
identified in a variety of loci (Li et al., 1999; Li et al., 2002).
Activation of a genomic domain by such control elements is
necessary for activation of individual genes in the cluster but
may not be sufficient. The second class of element comprises
the enhancers and promoters that decide the activity of
individual genes within a cluster. Finally, the third kind of
regulatory sequence is the boundary elements (also called
insulators) that separate gene clusters. These limit the range of
control of long-distance regulatory elements in the cluster, such
as LCRs, silencers and enhancers (Gerasimova and Corces,
2001; Labrador and Corces, 2002; Schedl and Broach, 2003;
West et al., 2002).
The best-studied gene cluster is the β-globin gene cluster in
man, mouse and chick (Levings and Bungert, 2002). Although
this cluster is exceptional, since the transcription rates in blood
cells are extremely high, it nevertheless has several of the
elements mentioned above. An LCR upstream of the cluster
switches the locus from a closed and inactive chromatin
configuration to a more open one, allowing transcriptional
activation (Festenstein and Kioussis, 2000; Li et al., 2002).
In addition, each of the genes has its own promoter, which
directs developmentally regulated expression of the gene.
Furthermore, boundary elements that flank the gene cluster
have been identified (Farrell et al., 2002). The example of the
β-globin cluster shows that cells regulate genes at different
hierarchical levels. Not all gene clusters are controlled in the
same way, however. In common with the β-globin cluster,
expression of the α-globin genes is developmentally
controlled. In contrast to the β-globin genes, the α-globin
cluster is in a typical euchromatic environment, i.e. one that is
gene rich and CG rich, early replicating and nuclease-sensitive
in non-haematopoietic cells (Brown et al., 2001). Moreover,
in human lymphocytes, which do not express haemoglobin,
the β-globin locus is associated with pericentromeric
Hierarchical genome function
heterochromatin, whereas the α-globin locus is not (Brown et
al., 2001). The α-globin cluster does not appear to switch
between a heterochromatic and a euchromatic state, but rather
has a permanently open conformation. Possibly, α-globin
genes are regulated exclusively at individual gene level, helped
by a locus-specific upstream regulatory element (Vyas et al.,
1992). The cell thus evidently uses different control
mechanisms for different genomic domains.
One approach to identify regulatory elements that control
gene expression at the level of gene clusters is to compare
genomic sequences of different organisms, assuming that
functional gene clusters and related regulatory elements tend
to be evolutionary conserved. Comparison of the human and
mouse sequences has already revealed a variety of conserved
non-coding sequences that are candidates for such regulatory
elements (Hardison, 2000; Mural et al., 2002; Shabalina et al.,
2001; Ureta-Vidal et al., 2003). An alternative route is the
identification of clusters of transcription-factor-binding sites
that are typically a few hundred base pairs in size (Berman et
al., 2002). Finally, once boundary elements can be identified
on the basis of nucleotide sequence alone, which is currently
not possible, their distribution in the genome will reveal
much about the one-dimensional functional organization of
eukaryotic genomes.
Versteeg and co-workers (Caron et al., 2001) have shown
that highly active genes tend to be organized in large clusters
(many megabases in size) on various chromosomes. Moreover,
housekeeping genes have been found to form several large
clusters in the mammalian genome (Lercher et al., 2002).
Surralles et al. (Surralles et al., 2002) have shown that
transcription-coupled DNA repair (TCR) is very prominent
in specific chromosomal domains. Since TCR is tightly
associated with active genes, it is likely that such domains
overlap those containing highly active genes. Given the size of
these clusters (many megabases per cluster, representing
hundreds of genes), they are unlikely to correlate with
individual regulatory units. Rather, this level of clustering
might be related to nuclear compartmentalization – for
instance, the creation of nuclear domains that are particularly
suited for high rates of gene expression and RNA processing
(see below).
The observations discussed above show that genes are
arranged far from randomly on the linear genome. We are
beginning to see the functional implications of this. A better
understanding of this level of genome function is essential if
we are to understand the orchestration of gene expression in
eukaryotes.
The chromatin level
In eukaryotic cells nuclear DNA is packed as chromatin
(Ridgway and Almouzni, 2001). The basic chromatin unit is
the nucleosome, consisting of about 150 base pairs of DNA
tightly wrapped 1.7 times around a protein octamer. The
octamer comprises two of each of the highly evolutionary
conserved core histones H2A, H2B, H3 and H4. Adjacent
nucleosomes are connected by 30-40 base pairs of linker DNA
that is more accessible than DNA in direct contact with the core
histones. Each chromosome consists of a single huge
nucleosomal fibre. Its 3D folding is dynamic rather than static.
For instance, the nucleosomal fibre is densely packed in
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metaphase chromosomes, whereas in interphase it is on
average about five times less compact (Manders et al., 2003).
Mounting evidence indicates that the folding of the
nucleosomal array in the interphase nucleus is an important
element in genome function. Chromatin can occur in various
structural and functional states. Transitions between chromatin
states are tightly linked to changes in gene activity (Eberharter
and Becker, 2002) and are causally related to changes in
covalent modification of core histone proteins (Strahl and Allis,
2000).
A considerable number of post-translational modifications
of core histones have been identified. Thirteen lysine residues
in the N-terminal tails of the four histone proteins can be
reversibly acetylated, three serine residues in histones H3 and
H4 can be phosphorylated, six lysine side-chains and three
arginine residues on histones H3 and H4 can be methylated,
and two residues in the C-terminal domains of histones H2A
and H2B can be ubiquitylated (Jenuwein and Allis, 2001;
Lachner et al., 2003). Most of these modifications are carried
out by enzymes that are recruited by sequence-specific DNAbinding proteins. Histone modifications specify functional
states of chromatin in a combinatorial manner (Turner, 2002).
For instance, hyperacetylation is related to transcriptionally
active and relatively open chromatin (Eberharter and Becker,
2002), whereas methylation of Lys9, Lys27 and Lys35 of
histone H3 is linked to transcriptional silencing and chromatin
compaction, often called heterochromatinization (Lachner
and Jenuwein, 2002). To make things more complicated,
methylation of histone H3 at Lys4 is associated with
transcriptional activity, (SantosRosa et al., 2002; Zegerman et
al., 2002). Together, this set of post-translational modifications
represents the histone code (Jenuwein and Allis, 2001; Strahl
and Allis, 2000; Turner, 2002), which adds an extra level of
information to the genome that is linked to DNA sequence only
indirectly. It acts as an epigenetic memory of what happened
to the cell lineage in the past, which is important in embryonic
development and cell differentiation (Turner, 2002).
Histone acetylation and phosphorylation are rapidly
reversible. The in vivo acetylation level of nucleosomes, and
therefore overall gene activity, has been shown to represent a
steady state, depending on the relative activities of histone
acetyltransferase (HAT) and histone deacetylase (HDAC)
enzymes (Im et al., 2002; KatanKhaykovich and Struhl, 2002).
Both types of enzyme can be recruited to specific sites in the
genome by sequence-specific transcription factors (Ng and
Bird, 2000; Roth et al., 2001). Interestingly, histone lysine
methylation is probably not reversible, since no demethylase
has been found so far, and the modification is chemically stable
(Bannister et al., 2002). Therefore, it seems that silencing by
histone methylation is reversed only by the slow process of
exchange of histone proteins in nucleosomes (Kimura and
Cook, 2001) or DNA replication. Also, specific factors that
locally speed up the release of methylated histones and allow
their replacement with other histone molecules might exist
(Ahmad and Henikoff, 2002).
One other chromatin modification that is closely related
to gene silencing is DNA methylation by DNA
methyltransferases (Geiman and Robertson, 2002). Coupling
of DNA methylation, histone deacetylation and methylation of
histone H3 Lys9 results in heterochromatinization and gene
silencing (Grandjean et al., 2001; Gregory et al., 2001b;
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Journal of Cell Science 116 (20)
Jackson et al., 2002; Johnson et al., 2002; Kouzarides, 2002;
Satoh et al., 2002; Soppe et al., 2002; Tamaru and Selker,
2001). Chemical modification of histones and DNA are
probably different aspects of one and the same regulatory
system.
Regulation of gene expression at the histone code level
creates a regulatory system that switches chromatin domains
between different functional states. Do other regulatory
mechanisms exist? One group of candidates is the multisubunit
ATP-dependent chromatin-remodelling machines (Langst and
Becker, 2001; Peterson, 2002). Members of this enzyme family
can rearrange nucleosomes in the chromatin fibres in an ATPdependent manner. The role of remodelling at the promoter
level, involving a small number of nucleosomes, has been
investigated in detail (Neely and Workman, 2002). There is
evidence that remodelling enzymes are also involved in the
unfolding of large chromatin domains (Peterson, 2003).
Interestingly, linker histones, which are often associated with
condensed inactive chromatin, are potent inhibitors of
chromatin remodelling, which creates a functional link
between the chromatin state and the remodelling machinery
(Horn et al., 2002). Remodelling enzymes also play a role in
chromatin condensation. Mutations in the Drosophila ISWI
gene, which encodes a member of the SWI/SNF ATPdependent remodelling family, produce a less compact male
X-chromosome (Corona et al., 2002). ISWI-mediated
remodelling is probably responsible for regular spacing of
nucleosomes on the chromatin fibre, allowing generation of
more tightly packed chromatin. These observations show that
switching chromatin between active and inactive states can be
controlled by different mechanisms – changes in histone
modification or remodelling. Both systems, however, do appear
to be tightly interlinked (e.g. Corona et al., 2002; Neely and
Workman, 2002).
All this adds to the classical regulatory mechanisms
provided by transcription factors. There is considerable
crosstalk between these two regulatory levels because at least
in part they use the same set of cis- and trans-acting
components. For instance, HAT activity is often recruited
through transcription factors that bind to promoters and/or
enhancers, often in combination with ATP-dependent
chromatin-remodelling enzymes (Gregory et al., 2001a;
Mizuguchi et al., 2001). In addition, many other genomic
regulatory elements act by recruiting enzymes involved in
chromatin modification. For instance, LCRs bind HATs (Ho et
al., 2002; Levings and Bungert, 2002), and silencing by
Polycomb group (PcG) proteins involves HDAC activity and
histone H3 Lys9 and Lys27 methylation (Cao et al., 2002;
Muller et al., 2002; Sewalt et al., 2002; Tie et al., 2001). These
histone modifications not only occur locally but can spread
along the chromatin fibre, thereby inducing a change in the
functional state of a complete chromatin domain containing
one or more genes (Fig. 1 and see below). This property, which
is discussed below, is likely to be a key element in the
regulation of complex genomes.
The relationship between the sequence level and the
chromatin level
It is a formidable task for a cell to coordinate the expression
of thousands of genes. In practice, for each genome a limited
number of useful gene expression repertoires exist, each
corresponding to a specific differentiation state. Although
precise numbers are lacking, it is reasonable to assume that in
each particular stably differentiated cell type, only a relatively
small fraction (10-30%) of genes can be transcribed at any
given time. The eukaryotic genome seems to be hard wired so
that only a limited number of stable states can exist, each
characterized by a specific repertoire of expressible genes.
How does the histone code allow the cell to orchestrate gene
activity? Histone-modifying enzyme activities are recruited by
sequence-specific transcription factors to the correct genomic
loci (Jenuwein and Allis, 2001; Turner, 2002). Somehow, the
histone modification state and, consequently, the functional
state of chromatin, seems to spread in cis along the chromatin
fibre (Fig. 1). Elegant studies of Noma et al. (Noma et al.,
2001b) on the 20 kb silent-mating-type locus of fission yeast
suggest that both the transcriptionally activated state
(characterized by histone acetylation and histone H3 Lys4
methylation) and the silenced state (characterized by histone
H3 Lys9 methylation) spread in this way. Inactivation of the
locus is initiated by a centromere-like repeat and spreads in
both directions until boundary elements are reached (Hall et
al., 2002). The hyperacetylated state of chromatin can also
spread along the chromatin fibre (Forsberg and Bresnick, 2001;
Ho et al., 2002). The molecular mechanism of spreading in cis
is not known, but may involve tracking of histone-modifying
enzymes along the chromatin fibre. Evidence indicates that
transcription by RNA polymerase I is involved in spreading of
the heterochromatin state of the rRNA gene cluster along the
chromatin fibre (Buck et al., 2002). Similarly, intergenic
transcription, as observed in the β-globin gene cluster and the
Hox gene cluster, by RNA polymerase II is probably involved
in changing the histone modification state and therefore the
functional state of the gene clusters (Plant et al., 2001; Rank
et al., 2002). LCRs are candidates for genomic sequence
elements that trigger switching of the functional state of
chromatin domains and may be starting points for intergenic
transcription (Gribnau et al., 2000; Ho et al., 2002; Plant et al.,
2001).
Boundary elements (insulators) constitute a currently only
operationally defined heterogeneous class of genomic element
and may act as boundaries of chromatin domains and at least
in some cases limit spreading along the chromatin fibre (Cuvier
et al., 2002; Labrador and Corces, 2002; Noma et al., 2001)
(Fig. 1). They block in cis the activating and inactivating effects
of long-range regulatory elements, such as enhancers, silencing
induced by PcG proteins and heterochromatin protein 1 (HP1)
(Van der Vlag et al., 2000; West et al., 2002), and LCRs (West
et al., 2002). Boundary elements delimit functional genomic
domains, such as the β-globin gene cluster and the fission yeast
mating-type cluster (West et al., 2002). Moreover, they are
able to shield transgenes from inhibitory position effects
(RecillasTarga et al., 2002).
There is thus increasing evidence that the linear genome is
functionally compartmentalized and that genomic domains
containing one or more genes are regulated independently.
Domain activation and inactivation are achieved through
recruitment of the relevant complement of histone-modifying
enzymes and a switch in functional state of a domain probably
involves spreading of the histone modification state along the
chromatin fibre until a boundary element is encountered.
Hierarchical genome function
The nuclear level
So far, we have treated the genome as a ‘simple’, linear coding
system in which regulatory elements act only in cis. In the cell
nucleus, however, chromatin is in a highly folded state that
brings together loci that are far apart on the linear genome and
may even be located on different chromosomes. It is likely that
regulation of genome function at the nuclear level can occur in
trans. Transvection is an example. Here, regulatory elements
that control expression of one allele (e.g. an enhancer)
functionally, and probably physically, interact with the
promoter of the allele on the homologous chromosome
(Duncan, 2002; Pirrotta, 1999; Wu and Morris, 1999).
Homologous pairing may be involved, providing a good
example of in trans interactions (Fung et al., 1998; Sass and
Henikoff, 1999). Our knowledge of molecular mechanisms of
genome function at this level is still very limited.
The interphase nucleus is functionally compartmentalized
(CarmoFonseca, 2002; Francastel et al., 2000). Many different
domains have been identified, each having a specific function
and macromolecular composition (Spector, 2001). There is
considerable evidence that the architecture of the nucleus is
closely related to genome function. The position of a gene
inside the nucleus is important: some areas are repressive,
whereas others promote or even boost transcription. For
instance, targeting of a gene to the periphery of the yeast cell
nucleus causes silencing (Andrulis et al., 1998). Repositioning
of genes to pericentromeric chromatin during lymphocyte
differentiation in mice correlates with epigenetic inactivation
of these loci, suggesting that contact with centromeric
heterochromatin results in spreading of the inactivated state in
trans (Brown et al., 1997; Fisher and Merkenschlager, 2002).
What DNA sequence elements and proteins control higherorder chromatin structure in relation to gene activity? Analysis
of position effects in transgene expression has provided some
insights (Mishra and Karch, 1999; Udvardy, 1999). Sequence
elements such as boundary elements (Udvardy, 1999), LCRs
(Li et al., 2002) and possibly certain types of enhancer
(Francastel et al., 1999) can overcome, in part, silencing owing
to association with heterochromatin in cis and/or in trans by
enforcing a functional state of a chromatin domain that allows
its genes to be transcribed (Dillon and Sabbattini, 2000).
A general problem in investigating the relationship between
nuclear organization and function is that we lack tools to
manipulate nuclear structure. For instance, we cannot
reposition at will individual genomic loci, induce changes in
local chromatin structure, or interfere with the positioning or
assembly of subnuclear domains and subsequently analyse
their effects on gene expression. Therefore, most results so far
have allowed us to observe correlations rather than causal
relationships. This is a major problem in this field. Evidently,
we first must identify the molecular components that determine
the different aspects of nuclear structure, including chromatin
structure and subnuclear domains, before we can interfere with
their function and analyse effects on gene expression. Several
attempts to tackle this problem are underway. The approaches
taken include interfering with the function of specific proteins
and biochemical isolation of specific nuclear domains and
subsequent identification of all their constituent proteins. These
experiments are revealing several aspects of nuclear
architecture that can be related to gene expression. Some of
them are addressed below.
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Chromosome territories
Individual chromosomes are readily visible only during
metaphase. In contrast, classical light and electron microscopy
of interphase nuclei do not reveal the position, size and
shape of chromosomes. In situ hybridization (ISH), using
chromosome-specific probes, has shown in many different cell
types and organisms that each interphase chromosome
occupies a unique, relatively compact, volume in the nucleus
(Cremer and Cremer, 2001). There is only limited
intermingling of chromatin from different chromosomes
(Visser et al., 2000).
What is the molecular basis for the formation of
chromosome territories? The simplest answer is that
chromosomes in interphase have decondensed only to a limited
extent after mitosis. This would preclude significant
intermingling of chromatin from different chromosomes
(Visser et al., 2000). In such a scenario, only chromosomal
stretches that are strongly decondensed can loop out and
intrude into other chromosome territories (Mahy et al., 2002a;
Volpi et al., 2000). The observation that the bulk of interphase
chromatin is only five times less condensed than in metaphase
chromosomes is consistent with this notion (Manders et al.,
2003).
Despite the relative compactness of their chromatin,
chromosome territories are open structures. Confocal light
microscopy and electron microscopy after thin sectioning
showed that there is a considerable volume of interchromatin
space inside chromosome territories (Cmarko et al., 1999;
Verschure et al., 2002; Verschure et al., 1999; Visser et al.,
2000). This is not a fixation artefact, because living cells in
which chromatin is labelled by incorporation of GFP-histone
H2B into chromatin show a spatial distribution of chromatin
similar to that in fixed cells (Hendzel et al., 2001; Politz et al.,
1999; Verschure et al., 1999). The interchromatin space
constitutes a highly convoluted set of channels, which allow
diffusion of macromolecular components through the nucleus,
including hnRNP particles (Politz et al., 1999). This
configuration is dynamic, since interphase chromatin is quite
mobile and shows constrained diffusion over distances up to a
few tens of a micrometer (Gasser, 2002). Transcription sites
are concentrated near the surface of compact chromatin
domains in the interchromatin space (Cmarko et al., 1999;
Fakan, 1994; Verschure et al., 1999). They probably represent
chromatin loops that extend into interchromatin space,
emanating from the compact chromatin domain (Fig. 1).
Interestingly, Cmarko et al. (Cmarko et al., 2003) have
presented evidence that PcG-silenced loci are located
predominantly in the same perichromatin area. Apparently,
transcriptionally active and silenced loci are physically close
together in the same subnuclear compartment, i.e. the
perichromatin domain.
The structure of interphase chromosomes in Arabidopsis has
been analysed in detail. Here, centromeric heterochromatin
acts as an organizing centre on which the gene-rich
chromosomal arms fold back to form multiple loops, forming
a rosette-like structure (Fransz et al., 2002). The much larger
mammalian chromosomes have a more complex structure and
contain more non-coding DNA. They might have a similar
structural organization, possibly having more than one
heterochromatin-organizing centre per chromosome and
forming multiple rosette structures.
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Journal of Cell Science 116 (20)
Large-scale chromatin structure and gene regulation
Transcriptional activation correlates with chromatin
decondensation (Fig. 1). This is particularly striking when an
array of genes is activated. The chromosomal fibre unfolds
extensively, occasionally looping out away from the body of
the chromosome territory (Mahy et al., 2002b; Tumbar et al.,
1999; Volpi et al., 2000; Ye et al., 2001). Several studies show
that chromatin decondensation alone is not sufficient for
transcriptional activation, however (Nye et al., 2002; Tumbar
et al., 1999; Volpi et al., 2000). This is consistent with the
notion that regulation at the chromatin domain level prepares
DNA for regulation at the level of individual genes, but does
not necessarily transcriptionally activate them.
Although the relationship between decondensation of
chromatin and transcriptional (pre)activation is wellestablished, our understanding of the process at the molecular
level is limited. What is chromatin decondensation? Obviously,
the average distance between the nucleosomes in a 3D
chromatin domain increases. Does this mean that non-covalent
interactions between nucleosomes in the compact structure are
disrupted? Possibly, specific proteins that are involved in
locking chromatin in its condensed state are released. This may
be true for HP1 during the transition from heterochromatin to
euchromatin. A pertinent question is what is the default state
of the nucleosomal fibre under the conditions that exist in the
interphase nucleus. Does chromatin (DNA plus core histones)
alone form an open structure that requires special factors to
compact, or does it self-assemble into a compact structure that
requires special molecules and/or chemical modifications to
open up? It is tempting to speculate that both are true, i.e.
chromatin needs special modifications/proteins to generate its
transcriptionally permissive state and others to stabilize its
inactive state. Nucleosomal arrays would thus exist in a labile
intermediate state if no other proteins are present and the
histones are unmodified. Indeed, chromatin is forced to
condense by specific histone modifications (e.g. methylation of
lysine 9 of histone H3) and subsequent binding of proteins,
such as HP1 (Grewal and Elgin, 2002; Turner, 2002). Other
chromatin-binding proteins and histone modifications,
however, may do the opposite, i.e. make chromatin structure
more open – for example, histone acetylation, methylation of
histone H3 on lysine 4 and ATP-dependent chromatin
remodelling (Memedula and Belmont, 2003).
Spatial arrangement of chromosome territories in the
nucleus
There seems to be no specific order in the arrangement of
chromosomes inside the eukaryotic nucleus, except that genedense chromosomes more often reside in the centre, whereas
gene-poor chromosomes reside more frequently in the
periphery (Cremer et al., 2001). This arrangement is
evolutionary conserved (Tanabe et al., 2002). Transcription
itself might thus be responsible for the observed radial
distribution. It is not clear whether chromosome territories
constitute a type of compartmentalization that is essential for
proper genome function. The fact that chromosomes can
exchange large fragments without impairing cellular function,
provided that no loss or gain of function is induced at the
chromosomal breakpoints, suggests that the radial organization
of chromosomes is not essential.
The link between the nuclear level and the
chromatin level
How is the 3D organization of the nucleus, and in particular its
chromatin, related to the 1D structure of the genome? And to
what extent is the spreading of active and inactive states of
chromatin, which is important in in cis regulation, also
important at the 3D level, i.e. in trans. There are indications
that in trans spreading of chromatin states does occur. For
instance, pairing of the Drosophila brown locus with a mutant
allele that contains a large block of heterochromatin results in
inactivation of the wild-type allele, which probably involves its
heterochromatinization. The inactive chromatin state may thus
be transmitted in trans (Csink and Henikoff, 1996; Dreesen et
al., 1991; Sass and Henikoff, 1999), which adds an additional
level of complexity to the dynamic organization of the
eukaryotic genome. Is the molecular mechanism of in trans
spreading different from spreading in cis? And what limits
spreading in trans in the 3D space of the nucleus? Could
insulators play a role, or should other mechanisms be invoked
that limit spreading in trans?
Another question concerns the relationship between
functional domains of the linear genome, i.e. gene clusters (see
above). Such domains may reflect chromatin loops attached at
their base to some kind of scaffold. It has been suggested that
a fibrous intranuclear network, the nuclear matrix, fulfils this
role. However, this concept is controversial (Nickerson, 2001;
Pederson, 2000). An alternative view is advocated by Corces
and co-workers (Labrador and Corces, 2002), who have
suggested that boundary elements delimit functional chromatin
domains in the nucleus by forming higher-order 3D structures.
Recently, Blanton et al. (Blanton et al., 2003) have produced
evidence that different insulator-binding proteins interact,
suggesting that the insulator-bounded domains form loops in
the nucleus. Finally, chromatin itself might form a compact
structure in the nucleus, from which chromatin loops emanate.
Such a model is consistent with the observation that
transcription takes place at the surface of relatively condensed
chromatin domains (Cmarko et al., 1999; Fakan, 1994;
Verschure et al., 1999). It is conceivable that intergenic, noncoding chromatin has this structural role.
Recently Versteeg and co-workers showed that genes that are
highly expressed in a large number of different human cell types
are strongly clustered on a number of chromosomes (Caron et
al., 2001). Such clusters are large, comprising hundreds of
genes present over many megabases. The clusters of highly
expressed genes not only contain housekeeping genes (Lercher
et al., 2002), but also loci expressed in specific cell types that,
if expressed, display high transcriptional activity. Some of these
clusters may correspond to chromatin that forms giant loops
bulging out of chromatin territories (Mahy et al., 2002a; Volpi
et al., 2000). For instance, the MHC locus on human
chromosome 6 represents a large cluster of highly transcribed
genes that forms a loop upon transcriptional activation (Volpi
et al., 2000). This indicates that inside the cell nucleus genes
that are transcribed at high rates form special domains or
compartments, creating a distinct link between the 1D
organization of the genome and the 3D structure of the nucleus.
Perspective
Whatever the link between nuclear architecture and regulation
Hierarchical genome function
of gene expression is, it is an important but still poorly
understood aspect of genome structure and function (O’Brien
et al., 2003). Understanding its molecular basis will be
essential if we want to understand the orchestration of gene
expression in eukaryotes.
What therefore are the major questions that we must answer
in order to begin to understand how the genome functions in
the confined space of the cell nucleus? The most prominent one
concerns the relationship between 3D chromatin structure and
function. We lack information about the static and dynamic 3D
arrangement of nucleosomes in different types of chromatin. It
is attractive to start from the idea that chromatin can adopt
a limited set of different spatial configurations reflecting
different functional states and that gene regulation in part is
due to the controlled switching between these defined states.
This process appears to be closely related to histone
modification and is ultimately encrypted in the DNA sequence
itself.
Chromatin structure must be studied in the living cell, since
it probably changes dramatically if the cell is disrupted. A
breakthrough has been the development by Belmont and coworkers of methods for GFP tagging of specific genomic sites
(Belmont, 2001). As the spatial resolution of light microscopy
is approaching the size of a few nucleosomes, there is hope that
detailed information about chromatin structure can be obtained
in the next few years (Esa et al., 2000; Kano et al., 2002).
We also need quantitative models of genome function that
link transitions between defined structural and functional
chromatin states to protein binding and histone modification.
Such models should constitute a critical guide for rational
experiments, whose results can be fed back into the model.
Finally, we urgently need tools to manipulate nuclear
organization. This should allow us to rearrange chromatin and
chromosomes and interfere with the assembly or function of
nuclear domains and subsequently establish the effects on gene
expression. Clearly, we first have to learn more about which
proteins and possibly RNA molecules are essential for nuclear
organization and chromatin structure. Subsequently, they can
become targets for experiments that interfere with nuclear
structure and function.
The authors acknowledge the financial support by several grants
from the biological branch (ALW) of the Dutch research council
NWO for research on nuclear structure and function.
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