REVIEWS
REPLICATION AND
TRANSCRIPTION: SHAPING THE
LANDSCAPE OF THE GENOME
Lyubomira Chakalova, Emmanuel Debrand, Jennifer A. Mitchell,
Cameron S. Osborne and Peter Fraser
Abstract | As the relationship between nuclear structure and function begins to unfold,
a picture is emerging of a dynamic landscape that is centred on the two main processes
that execute the regulated use and propagation of the genome. Rather than being
subservient enzymatic activities, the replication and transcriptional machineries provide
potent forces that organize the genome in three-dimensional nuclear space. Their activities
provide opportunities for epigenetic changes that are required for differentiation and
development. In addition, they impose physical constraints on the genome that might help
to shape its evolution.
Laboratory of Chromatin
and Gene Expression,
The Babraham Institute,
Babraham Research
Campus, Cambridge
CB2 4AT, United Kingdom.
Correspondence to P.F.
e-mail:
[email protected]
doi:10.1038/nrg1673
Published online 10 August 2005
NATURE REVIEWS | GENETICS
Based on studies of a small number of individual genes,
it has been known for decades that there is a correlation
between transcription and DNA replication in early
S phase. Active genes tend to replicate early in S phase
whereas inactive genes replicate late1,2. Replication
imposes the need to re-establish epigenetic information
on both daughter fibres of newly replicated chromatin
so that cells can continue their specific gene-expression
programmes for the remainder of the current cell cycle,
and inherit or remember those patterns in the next cell
cycle. This has led to the speculation that chromatin
assembly at replication forks could specify geneexpression states (see REF. 3 and references therein).
In particular, it was proposed that early replication
could specify permissive chromatin states that allow
gene expression, whereas late replication could specify
heterochromatin assembly at silent regions. Recent
large-scale studies have confirmed a strong correlation
between early DNA replication and gene expression,
fostering the link between transcription and replication
timing4,5. However, these studies also uncovered many
expressed genes that replicate late and many inactive
genes that replicate early. So what is the link between
transcription and replication timing? An appreciation
of the structure of the genome and its organization in
the nucleus relative to the transcription and replication
machineries, coupled with revealing findings on the
increased complexity of the transcriptome, indicates a
model that could explain the interplay between these
processes.
Transcription has long been thought to be the
primary regulatory step in controlling most geneexpression programmes in differentiation and development. Much attention over the years has focused on
the control of mRNA expression, which is the product of protein-coding genes. However, recent work
shows that mRNA, and in particular polyadenylated
mRNA, is the least complex RNA population in cells6.
Analysis of the human transcriptome has shown that
transcription occurs over much wider areas of the
genome than can be accounted for by protein-coding
genes6,7. This realization, coupled with an appreciation that transcription is highly compartmentalized
within the nucleus, indicates that the transcriptional
machinery has a central role in the organization of
the genome.
In this review we focus on the spatial and temporal
control of DNA replication and transcription, pointing out how these processes both reflect and shape
the nuclear landscape of the genome. We relate the
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a
b
c
Figure 1 | Patterns of replication foci in S phase cells. The images show pulse-labelled,
newly replicated DNA. The three nuclei, from left to right, show patterns that are typical for
early, mid and late S phase, in corresponding order. a | In early S phase, numerous small
replication foci are distributed throughout the nuclear interior. b | In mid S phase, replication
occurs mostly at the nuclear periphery and in nucleolar regions. c | In late S phase, a small
number of large, irregularly shaped replication foci localize to peripheral or internal
heterochromatic domains. Images courtesy of D. Dimitrova at the Center for Single
Molecule Biophysics, State University of New York.
observed genomic characteristics of highly transcribed,
early replicating and late-replicating sequences to the
organization of genes in the genome. We also propose
that the chromatin disruption and assembly that occurs
during replication and transcription might provide a
window of opportunity to alter epigenetic modifications and gene-expression states. We then relate the
results of recent studies of widespread transcriptional
activity across the genome to the nuclear organization
of transcription, and suggest that the transcriptional
machinery might be a powerful force that organizes the
physical arrangement of the genome in the nucleus and
thereby influences genome evolution. Specific aspects of
the genomic landscape outside the focus of this review,
such as heterochromatin formation and organization;
histone modification; chromatin remodelling; and the
rich and detailed biochemistry of these processes can be
found in the excellent reviews in REFS 810.
Replicating the genome
NUCLEAR LAMINA
A scaffold of proteins — mainly
lamin A/C and B — that are
predominantly found in the
nuclear periphery associated
with the inner surface of the
nuclear membrane.
NUCLEOLUS
A highly organized nuclear
organelle that is the site of
ribosomal RNA processing and
ribosome assembly.
NUCLEAR MATRIX
A three-dimensional
filamentous protein network in
the nucleus that remains intact
after high salt extraction.
LONG INTERSPERSED NUCLEAR
ELEMENTS
A class of repeat sequences.
670 | SEPTEMBER 2005
Proliferating cells duplicate their nuclear DNA before
each cell division during S phase of the cell cycle. Despite
the large size of metazoan genomes, replication is
accomplished in a matter of hours owing to the fact that
genomes are divided into thousands of individual replication units or replicons, with 10–15% simultaneously
active at any given time during S phase11. It is crucial
that each DNA sequence is replicated only once per cell
cycle. Therefore it is imperative that replication is tightly
controlled12. Pulse-labelling and direct visualization of
newly synthesized DNA reveals that DNA synthesis is
carried out at specialized replication sites or replication factories, each of which is co-occupied by several
active replicons13–15. Throughout S phase, these factories
assemble dynamically (FIG. 1). Replicons that are located
in their immediate vicinity are temporarily recruited to
the synthetic sub-compartment and, eventually, newly
synthesized DNA moves back to chromatin-rich regions,
away from its replication site15,16. Live cell studies confirm that no large-scale movements of chromatin take
place in S phase, but rather chromatin domains undergo
local rearrangements17 BOX 1.
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On completion of replication within local replicons, synthetic sites disassemble and new factories
form in the immediate vicinity in three-dimensional
space 14,17,18 . This pattern is clonally inherited, so
that sequences that replicate synchronously in one
S phase will replicate synchronously in the next13,14,17.
Chromatin domains that are replicated together
remain associated throughout the cell cycle, which
has led to the suggestion that they represent stable
chromosomal units14. Although the precise molecular
forces that sustain these structural units are largely
unknown, it is fair to presume the involvement of
interactions between chromatin domains and underlying nuclear structures such as the NUCLEAR LAMINA,
19–21
NUCLEOLUS and NUCLEAR MATRIX
.
DNA replication is also controlled temporally.
Recent genome-wide studies in Drosophila melanogaster
and humans have shown a correlation between early
replication timing and an increased probability of gene
expression4,5,22. The correspondence between active
regions and early replication, and inactive regions and
late replication, is not absolute. There are exceptions in
each case, but in general GC-rich, gene-dense regions
tend to replicate early, whereas AT-rich regions that
are usually gene-poor and have a high LONG INTERSPERSED
NUCLEAR ELEMENT (LINE) content replicate late. In one
case in which replication timing of a limited number
of specific genes was assessed in different cell types,
the replication-timing pattern was remarkably similar
from one cell type to the next23. Most genes did not
change their replication timing in association with
expression changes23. A recent large-scale study in
D. melanogaster has shown a surprising similarity in
Box 1 | Chromatin dynamics
Chromatin loci are highly mobile but their motion is
restricted within confined volumes20,89,126. In short
time intervals (1–2 sec), a fluorescently labelled
locus oscillates within a volume with an average
radius of ~0.3 µm, which is indicative of local
tethering. This is consistent with a model of
Brownian motion that is constrained by interactions
with immobile nuclear structures. However, when
observations are made over longer periods of time
(between 30 sec and several minutes), it becomes
obvious that genomic loci can make much larger,
long-range movements (up to 1.5 µm in 1 min
and up to 3 µm in 10 min). This indicates that
short-range confinement can be transient, perhaps
resulting from short-lived associations (see
supplementary information S1 (movie)).
Long-range mobility is probably constrained by
chromosome structure. Individual chromosomes are
non-randomly arranged within the nucleus and
occupy discrete territories that seem to be almost
immobile through most of the interphase127. Although
chromosomes are relatively static, individual
chromatin domains undergo constant Brownian
motions and can extend far beyond the edges of their
chromosome territory.
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the replication timing of late-replicating sequences
in two unrelated cell types24. However, a recent assessment of replication timing of 54 genes expressed at
different stages during embryonic stem-cell differentiation into neural precursors showed that replication timing can change in association with expression changes25,
although the timing changes seem to be restricted to
genes in AT-rich regions. Genes in GC-rich regions did
not change their replication timing during differentiation, regardless of changes in gene expression. These
results indicate that genomic context and organization
is important in defining replication timing, rather than
individual gene-activity states BOX 2.
Although GC content, gene density, nuclear position and transcriptional activity all strongly co-correlate
with replication timing, the causative nature of these
interrelationships is still a matter of debate and investigation. Considerable attention has focused on the
possibility that differential replication timing could
specify alternative chromatin assembly fates and
thereby have a role in controlling gene expression.
For example, early replicating sequences could use a
chromatin assembly pathway, resulting in a structure
that is permissive for gene expression, whereas latereplicating sequences could use machinery that would
result in a repressive or heterochromatic assembly.
There is evidence that repressive histone-modifying
activities and chromatin-remodelling complexes are
targeted to late-replication sites26–29, which suggests a
plausible model for maintaining repressive chromatin
states. Complementary evidence for early replicating
sites is noticeably lacking.
A window of opportunity
REPLICATION ORIGINS
DNA sequences at which DNA
replication is initiated.
NATURE REVIEWS | GENETICS
Fisher and Mechali found that retinoic-acid induction
of HoxB (homeobox B cluster) expression in P19 cells
occurred during, and required, S-phase progression30.
This led to a suggestion that S phase or DNA replication might provide a window of opportunity for geneexpression patterns to be changed. However, because
the HoxB cluster replicates early in both stimulated
and unstimulated P19 cells, something other than early
replication must also be necessary for induction.
Replication timing is probably determined by epigenetic information and chromatin structure rather
than by the expression of individual genes per se.
This is consistent with many of the observed correlations between replication timing, genomic context
and gene activity3,22,31,32. Interestingly, the replicationtiming programme in mammalian cells is established
early in G1 phase, concurrent with the post-mitotic
repositioning of chromatin domains, which indicates
a close link between nuclear positioning and replication timing33. This occurs before, and independently of, the specification of REPLICATION ORIGINS33–36. It
is unlikely that every gene has its own replication
origin — genes in gene-dense regions co-replicate
with their neighbours regardless of their individual
expression states.
The tissue-specific human α-globin and β-globin
clusters represent two opposite examples of the
Box 2 | Genome organization
GC content, repeat character and gene expression have
all been correlated with organization of the genome.
Genes are not randomly distributed across the genome
and detailed analysis of transcriptome maps shows
significant clustering of highly expressed genes, with
patterns that are remarkably similar among many cell
or tissue types128. The most highly expressed genes tend
to be clustered in genomic regions called RIDGEs
(regions of increased gene expression). RIDGEs are
characteristically GC-rich, have high gene density and
low LINE (long interspersed nuclear element) content.
Subdividing transcriptome data into tissue-specific
versus widely expressed genes and ignoring tandemly
duplicated genes reveals that tissue-specific genes
in general do not cluster129. The observed clustering
of genes with high-expression rates is the result of
multi-megabase size regions that contain a high
density of housekeeping genes129. Although individual
gene-expression levels change from tissue to tissue,
the global expression profiles remain similar.
Tissue-specific genes are often interspersed among
the widely expressed genes in RIDGEs130.
ANTIRIDGEs, regions of low gene density, and high
AT and LINE content have also been described; they
contain mainly weakly expressed genes130.
dissociation between replication timing and gene
expression. The α-globin genes are embedded in a
large cluster of widely expressed genes on chromosome 16. They are in a nuclease-sensitive conformation and replicate early in all cell types37–39, although
they are expressed only in erythroid cells. On the
other hand, the β-globin genes on chromosome 11
are embedded in a large cluster of olfactory receptor
genes, which are only expressed in nasal epithelium.
The β-locus is in a closed chromatin conformation
and replicates late in nearly all cell types, except erythroid cells in which it replicates early, in association
with chromatin opening40. The neighbouring genes in
the flanking olfactory receptor gene cluster also switch
to early replication in erythroid cells even though they
are not expressed41. Clearly, early replication does not
itself specify gene expression, but replication might
still have a role in changing gene-expression states by
transiently disrupting chromatin.
Recent studies indicate that transcription is also
highly disruptive to chromatin. The old idea that
chromatin is repressive to transcription came from
in vitro studies of purified components showing that
naked DNA is transcribed much more efficiently than
nucleosomal templates42. In vivo transcription occurs
exclusively on chromatin templates. Much recent
progress has been made in identifying and characterizing various components and complexes that aid the
passage of the transcriptional machinery 43. We now
know that transcription disrupts chromatin through
partial disassembly of the underlying nucleosomes,
followed by rapid reassembly in the wake of the
transcriptional machinery 44,45.
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Box 3 | Non-genic transcripts
Although it is difficult to define universally, at a minimal sequence level a gene has
traditionally been thought of as the region between the transcription start-site and
the polyadenylation site. This includes exons as well as the intervening intron
sequences, which on average account for about 95% of a gene’s length. During
transcription or immediately after, introns are excised from primary transcripts and
degraded as part of the RNA splicing process. The resultant mRNA molecules —
which consist of the joined exons, polyadenylated at their 3′ ends — are transported
to the cytoplasm. Although most known genes encode proteins, some have no
protein-coding potential and might produce functional non-coding RNA molecules
such as the X-inactive specific transcript (Xist) and antisense Igf2r (Air) RNAs.
Surprisingly, a recent survey of cytoplasmic poly(A)+ RNA using oligonucleotide
arrays that covered the non-repetitive sequences of two human chromosomes found
that ten times as much sequence was present in the cytoplasm as can be accounted for
by the known or predicted exons7. Most of these novel RNAs had low coding
potential, and were present at low levels. Some seemed to arise from genomic
sequences that are near to or overlapping exon sequences, often in the antisense
direction, whereas many more came from unannotated or non-genic regions,
indicating that a significantly larger proportion of the genome is transcribed than
was previously thought. So there might be more genes than currently predicted, but
there is also clear evidence for a considerable amount of non-genic transcription.
Similar to DNA replication, transient chromatin
disassembly during transcription might represent an
opportunity for change. Polycomb response elements,
which bind repressive POLYCOMB GROUP (PcG) complexes,
mediate the inheritance of silent chromatin states.
However, transcription through a Polycomb response
element results in the resetting of this epigenetic
memory and promotes formation of an active chromatin structure46–51. Moreover, recent work indicates
that transcription triggers histone turnover and the
deposition of the variant H3.3 histone in transcribed
regions50,52,53. H3.3 is highly enriched for modifications
that are associated with active chromatin54, which is
consistent with a role in setting up and maintaining an
active epigenetic state.
Transcribing the genome
We normally think of transcription as being exclusive to genes, but it now seems likely that much, if
not most, transcribed sequence in higher eukaryotic
genomes lies outside the areas that we recognize as
genes6 BOX 3.
A common proposal has been that these noncoding RNA (ncRNA) transcripts are the result of
‘leaky’ transcription. However, large-scale studies
have revealed evidence for their precise regulation55,56.
Several suggestions have been made about the function of these transcripts, including the suggestion that
they are involved in the regulation of specific gene
expression57,58. Recent evidence clearly indicates that
the process of transcription itself can have a regulatory role50,51,59–61 by altering chromatin conformation
or the association of regulatory factors with downstream control elements. The fact that most nuclear
transcripts never enter the cytoplasm6 indicates the
possibility that, in some cases, the ncRNAs that are
produced might be fortuitously processed by-products.
For some loci, these so-called intergenic transcripts
seem to delineate domains of modified chromatin that
surround active genes and their remote regulatory
elements62–67. Intergenic transcripts can be sense or
antisense66, and the sequences that encode them frequently overlap with nearby target genes68–71; their
expression precedes or is co-regulated with that of
their neighbouring genes. Detailed characterization
of several loci has revealed extensive intergenic transcription, and the list of loci for which this has been
observed is growing rapidly, although the precise role
of intergenic transcription is still unclear. The growing repertoire of chromatin remodelling and histonemodifying factors that are associated with the
elongating form of RNA polymerase II (PolII) indicates
a role in maintaining domains of modified chromatin,
and mounting evidence supports this43,72–76.
Transcription compartmentalization
2µm
POLYCOMB GROUP
A class of proteins — originally
described in Drosophila
melanogaster — that maintain
the stable and heritable
repression of several genes,
including the homeotic genes.
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Figure 2 | Transcription factories. The images show RNA
immuno-FISH (fluorescence in situ hybridization) in a mouse
erythroid cell. RNA FISH for haemoglobin β-chain complex
(Hbb) (red) and erythroid associated factor (Eraf ) (green)
genes and immunofluorescent detection of RNA polymerase II
(PolII) (blue) is shown. Hbb and Eraf are located on the distal
third of mouse chromosome 7, separated by 25 Mb. The
scale bar indicates 2 µm.
| VOLUME 6
Transcription is highly compartmentalized in mammalian nuclei77–81. The classic example is the segregation of RNA polymerase I complexes to the nucleolus,
where rRNA genes are transcribed. Similarly, nascent
transcription by PolII is not homogeneously distributed throughout the nucleoplasm, but occurs at highly
enriched PolII foci, known as transcription factories,
which contain most of the hyperphosphorylated,
elongating form of PolII REFS 8284 (FIG. 2). There are
fewer transcription factories than there are active genes
and other transcription units in the nucleus, which has
led to the prediction that more than one active gene is
transcribed in each factory78,82. Recent results show that
actively transcribed genes that are separated by up to
40 Mb of chromosomal sequence frequently co-localize
in the same transcription factory85. Interestingly, RNA
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Inactive (Hbb) gene
Classical
enhancer
activity
Unknown function
Intergenic
promoter activity
Insulator activity
Active (Hbb)
genes
Olfactory receptor
genes
Figure 3 | A chromatin hub. A hub that is formed by
long-range interactions between the haemoglobin β-chain
complex (Hbb) genes and the locus control region is shown.
Transcriptional activation involves physical association of
genes and their regulatory elements. This arrangement
could increase the local concentration of trans-acting
transcription factors by spatial clustering of binding sites for
such factors95,96,105. The coloured circles represent DNaseI
hypersensitive sites.
fluorescence in situ hybridization (FISH) analysis of
several genes at the single-gene level showed that most
‘active’ genes are not continuously transcribed but seem
to go through transcription cycles, spending more time
in the off state than in the on state85. This confirms
earlier studies86–88 and indicates that although related
cell types might express an identical set of genes at the
mRNA level, no two cells will have identical subsets
of actively transcribed genes at any given moment.
So turning on a gene seems to be unlike a switch that
gets turned on and stays on until switched off. Gene
transcription occurs in pulses at a certain frequency
that might serve to regulate, in part, the expression
level of a particular gene across a population of cells.
Modulation of expression might occur by changing the
probability that a gene is transcribed, thereby resulting
in an altered frequency or duration of ‘on time’ relative
to ‘off time’.
Osborne et al.85 showed that actively transcribed
genes co-localize with transcription factories, whereas
identical, temporarily non-transcribed alleles, which
can often be in the same cell, do not. Therefore, the on
state correlates with factory occupancy and the off state
with relocation away from factories. This indicates that
dynamic associations with factories underlie the binary
NATURE REVIEWS | GENETICS
oscillations in the transcriptional activity of genes and
raises the possibility of regulating genes by controlling
chromatin mobility89. The specific nuclear repositioning of genes has been correlated with transcriptional
activation, silencing and replication timing90–92. Two
recent studies highlight the extent and precision of
nuclear repositioning of active genes93,94. Chambeyron
and Bickmore compared the positions of two closely
linked homeobox (Hox) genes — one active, the
other inactive — relative to their chromosome territory93. They observed repositioning of the active
gene up to 1 µm away from the inactive gene and
its chromosome territory, indicating that chromatin
decondensation and increased mobility occur with
expression. The second study, by Zink et al.94, investigated the nuclear organization of the cystic fibrosis
transmembrane conductance regulator gene (CFTR)
in relation to nearby flanking genes. They showed
that CFTR adopted a more interior position when
expressed, although the inactive flanking genes remained
tethered to heterochromatin at the nuclear periphery.
Their experiments indicated that nuclear positioning is
controlled at the level of individual genes and is dependent on transcriptional activity.
Transcription and higher-order structures
The link between higher-order chromatin structures
and transcription has recently become clear. The mouse
haemoglobin β-chain complex (Hbb) genes and their
distal regulatory elements — the locus control region
(LCR), located more than 50 kb away — engage in
specific higher-order, ‘loop’ structures during transcription95,96. Loop structures have also been detected
in imprinted97,98 and cytokine99 clusters, and between
chromatin boundary elements100. It is notable that only
one of the nine distal regulatory elements that seem
to cluster around the active Hbb gene is known to be
a ‘classical’ enhancer. The other sequence elements
involved have insulator-like properties101, intergenic
promoter activity102–104 or unknown functions. They
coalesce into what has been called an active chromatin hub 105,106 (FIG. 3). Although long-range loops
are undoubtedly formed, several questions remain.
For example, how is specificity of contact controlled
between demonstrably promiscuous regulatory elements and their intended target genes, which could be
a megabase or more away107–109? Diffusion coefficients
calculated for chromatin fall within a range that would
allow for long-range interactions89, such as associations
between genes and regulatory elements. In theory, two
regions positioned within 1 µm from each other would
have a high probability of coming into contact within
seconds, but these calculations do not take into account
the fact that there might be hundreds of other eligible
genes nearby. Are there mechanisms beyond simple
diffusion that could promote the specific assembly
of distal elements over large distances? Finally, is an
organized cluster of regulatory elements sufficient to
create a transcription site, or do these complexes allow
entry to, or stabilize, associations with pre-assembled
factories?
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LCR transcript
Inactive gene
3′ Enhancer
intergenic transcript
Boundary
terminator
3′ Enhancer
elements
LCR
elements
Intergenic transcript
Chromatin loop
emerges
Antisense intergenic transcript
Bi-directional
non-coding RNA
(intergenic) promoter
Potentiated
gene with distal
3′ enhancer elements
Figure 4 | Organizer factory. Clustered RNA polymerase II motor proteins organize genes and distal regulatory elements
by using non-coding intergenic transcription. Rather than polymerases sliding along chromatin, chromatin moves through
the factory that is powered by the energy released by the hydrolysis of nucleotide triphosphates during elongating RNA
synthesis. Black arrows, gene promoters; blue arrows, direction of chromatin movement; LCR, locus control region; red
arrows, intergenic promoters.
Factories as organizers
The fact that different genes frequently co-occupy the
same factory provides strong evidence that genes do
not assemble their own transcription sites de novo
when they become active, but instead migrate to preassembled transcription sites85. The available data
indicate that transcription factories are metastable
associations that might assemble on an underlying
scaffold or nuclear matrix110–112. A stable factory implies
that genes or transcription units would essentially be
pulled through a factory113, rather than polymerases
moving along the chromatin fibre, as is commonly
believed (FIG. 4). It has been shown that prokaryotic
RNA polymerase is a powerful motor protein, which
can generate forces that are far in excess of other known
ATPases114,115. The tractor forces derived from efficient
conversion of the free-energy produced as a result of
nucleotide hydrolysis during intergenic transcript elongation could serve to ratchet distal regulatory elements
and their target genes into a common factory, thereby
facilitating the formation of long-range cis interactions,
rather than trans associations116. In such a model, large
distances between long-range enhancers and their target genes would not be an insurmountable problem. It
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was not long ago that even 50 kb seemed an unreasonably large distance for a long-range enhancer to make
a complex with its target gene in a specific regulatory
interaction95,96. Today such interactions must be considered as medium-range, as there is clear evidence for
associations that take place over several megabases85,117.
Once the loop is established the enhancer gene complex might help to stabilize a productive association
with a factory, resulting in enhanced gene transcription (re-initiation) or more efficient elongation118, or
both (FIG. 5). The finding that approximately 15% of the
genome is transcribed6 — although probably not all
at once — indicates that an extraordinarily large part
of the genome passes through the limited number of
transcription factories in a cell nucleus85. This must
have a profound effect on the nuclear organization of
the genome.
The transcription–replication interface
A DNA sequence that is being used as a template for
replication temporarily disengages RNA polymerases
and ceases transcription119, although global nuclear
transcription continues uninterrupted throughout
S phase. This requires precise coordination between
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early as bystanders. Whether these genes replicate early
from origins that are in neighbouring active domains
or use a local origin that becomes active owing to the
increased local concentration of replication machinery
in the factory remains to be determined. Chromatin
domain structure does not always respect the boundaries of gene-expression domains, as inactive genes can
be decondensed as a result of chromatin opening at
neighbouring genes109. This would explain both the
link between active genes and early replication, and
the characteristic nuclear distribution of replication
forks at any given time in S phase that grossly reflects
the compartmentalization of active and inactive
chromatin in the nucleus.
Genes
in trans
Implications for genome evolution
Distal genes
in cis
LCR–gene
complex
Transcription
factory
Figure 5 | Transcription factory model. In this model, genes extend out of their chromosome
territories in cis and trans to access a shared transcription factory. Coloured circles represent
DNA binding factors. LCR, locus control region.
transcription and replication at the level of individual
sequences, particularly during early S phase when
most active genes are replicated. How is this achieved?
Presumably a sequence that is engaged in transcription, which is due to be replicated, would first need to
dissociate from its transcription factory and relocate
to a nearby replication factory. There is evidence for
little or no overlap between replication and transcription sites120–122. Once replication is complete, there is
presumably a need for epigenetic modifications to
be re-established on the daughter strands. Will this be
accomplished at the replication site? Or will potentially active regions be reeled back into transcription
factories by intergenic transcription? Interestingly, in
the case of the β-globin genes, a pulse of intergenic
transcription occurs just after replication in early
S phase63.
To return to the question we posed at the beginning: what is the link between transcription and early
replication? We propose that early replication sites
that are present at the beginning of S phase form at
the most accessible replication origins. These will be
origins of replication, which are near active genes,
that will be clustered around transcription sites in the
nucleus. Interspersed between these active genes will
be some closely linked inactive genes that will replicate
NATURE REVIEWS | GENETICS
Transcription and replication are more than just things
that happen to the genome. They might represent
opportunities to change gene-expression programmes;
insert variant histones or modify existing ones; foster
higher-order structures; and organize the genome into
active zones in the nucleus. What influence might this
have on the arrangement of genes in the underlying
DNA sequence?
This functional nuclear organization could
be expected to impose selective pressures on the
organization of genes and regulatory elements at
the primary sequence level. Unlike protein-coding
regions, where selection acts at the codon level,
pressures on genome organization are probably
exerted at the level of higher-order chromatin and
chromosome structure, and the functional organization of the nucleus. The clustering of genes around
functional sites in the nucleus might be the selective
pressure that is responsible for the observed clustering of highly expressed genes in the genome. Each
potentiated gene in a local cluster of highly expressed
genes would have a greater chance of dynamically
interacting with a transcription factory, owing to
the fact that the entire region might remain tethered
to the factory through the engagement of one or
more local transcription units in a self-organizing123
transcriptional ‘frenzy’. Conversely, the discovery that
weakly expressed genes and genes that are involved in
the control of development and differentiation tend to
lie in relatively gene-poor regions of the genome might
reflect their need for finely controlled expression.
Such genes would do well to stay away from trancriptional hubbubs, which could result in inappropriately
high levels of expression. These types of gene might
also be expected to have independent control of
their chromatin environments and might therefore
be expected to change their replication timing in
association with expression124.
Concluding comments
The links between structure and function in the nuclear
landscape of the genome grow ever stronger. For
example, histones, which were once considered to be
mere structural units of chromatin, are now generally recognized as principal reservoirs of epigenetic
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Acknowledgements
The authors would like to thank A. Horton, P. Varga-Wiesz,
W. Reik and A. Corcoran for helpful discussions. We are also
grateful to S. Gasser for the yeast chromatin dynamics movie
and D. Dimitrova for DNA replication micrographs. J.A.M. is
supported by a Natural Sciences and Engineering Research
Council of Canada postdoctoral fellowship. P.F. is a Senior
Fellow of the Medical Research Council (MRC) and is supported
by the MRC and Biotechnology and Biological Sciences
Research Council, UK.
Competing interests statement
The authors declare no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
Air | CFTR | Eraf | Hbb | HoxB | Xist
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Author biographies
Lyubomira Chakalova obtained her Ph.D. at the Institute of Molecular
Biology in Sofia, Bulgaria, where she studied global DNA repair patterns in mammalian cells. She is currently a postdoctoral associate in
the Laboratory of Chromatin and Gene Expression at The Babraham
Institute, Cambridge, UK.
Emmanuel Debrand obtained his Ph.D. at the Pasteur Institute in
Paris, France, where he worked on the mechanism of X-chromosome
inactivation in the mouse. He is currently a postdoctoral researcher in
the Laboratory of Chromatin and Gene Expression at The Babraham
Institute.
Jennifer Mitchell received her Ph.D. from the University of Toronto,
Canada, on the role of AP-1 transcription factors during pregnancy. She is currently a postdoctoral associate in the Laboratory of
Chromatin and Gene Expression at The Babraham Institute.
ries that are shared with other actively transcribed genes.
• In higher eukaryotes approximately 15% of the genome is transcribed.
Most transcribed genomic regions are intergenic sequences.
• The finding that most of the genome is transcribed indicates that
transcription factories are principal focal points for the nuclear
organization of the genome.
• The clustering of genes around functional sites in the nucleus might
impose selective pressures on the organization of genes and regulatory elements at the primary sequence level, thereby contributing to
the observed clustering of highly expressed genes in the genome.
Online links
Entrez:
Air
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retr
ieve&dopt=Graphics&list_uids=104103
Cameron Osborne obtained his B.Sc. in biology at the Simon Fraser
University in Vancouver, Canada, and Ph.D. at the University of
Adelaide, Australia, studying the regulation of cytokine genes. He
was a postdoctoral associate at the Hospital for Sick Children in
Toronto, developing retroviral vectors for gene therapy. He is currently
a postdoctoral associate in the Laboratory of Chromatin and Gene
Expression at The Babraham Institute.
CFTR
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retr
ieve&dopt=Graphics&list_uids=1080
Peter Fraser did his graduate research at the Wistar Institute in
Philadelphia, USA, and obtained his Ph.D. from the University of
Pennsylvania, USA, studying coordinate control of gene expression.
As a postdoctoral researcher he studied β-globin gene transcription
at the National Institute for Medical Research in Mill Hill, London,
UK. He continued this work as a faculty member of the Erasmus
University Medical Center in Rotterdam, The Netherlands. He is
currently a Senior Fellow of the Medical Research Council, UK, and
Head of the Laboratory of Chromatin and Gene Expression at The
Babraham Institute, investigating chromatin, epigenetics and nuclear
organization of the genome.
Hbb
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retr
ieve&dopt=Graphics&list_uids=15127
Online summary
• DNA replication in mammalian nuclei is carried out in specialized structures that are known as replication factories. Throughout
S phase, these factories assemble dynamically, and replicons that are
located in their vicinity are recruited to the replication sub-compartment.
• GC-rich, gene-dense regions tend to replicate early in S phase,
whereas AT-rich regions that are usually gene-poor replicate late.
In many cases, expressed genes replicate early in S phase and silent
genes replicate late, but there are many exceptions to this rule.
• Early replication timing is probably determined by epigenetic
information and chromatin structure rather than by individual
gene expression per se. Gene expression does not seem to be a direct
consequence of early replication.
• Transcription is highly compartmentalized in mammalian nuclei.
Nascent transcription by RNA polymerase II occurs in foci that are
known as transcription factories. Cells contain very few transcription factories compared with the number of active genes.
• Most ‘active’ genes are not continuously transcribed, but undergo
transcription cycles. Modulation of expression might occur by
changing the frequency or duration of the ‘on’ state versus the ‘off ’
state.
• On activation, genes migrate to pre-assembled transcription facto-
Eraf
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retr
ieve&dopt=Graphics&list_uids=170812
HoxB
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retr
ieve&dopt=Graphics&list_uids=15406
Xist
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene&cmd=Retr
ieve&dopt=Graphics&list_uids=213742