Human Molecular Genetics 2005 14(Review Issue 1):R101-R111; doi:10.1093/hmg/ddi104
© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org
Remote control of gene transcription
Adam G. West1,* and
Peter Fraser2
1Division of Cancer Sciences and Molecular Pathology, University of Glasgow, Western Infirmary, Glasgow G11 6NT, UK and
2Laboratory of Chromatin and Gene Expression, The Babraham Institute, Babraham Research Campus, Cambridge CB2 4AT, UK
* To whom correspondence should be addressed. Tel: +44 1412112974; Fax: +44 1413372494; Email: a.west{at}clinmed.gla.ac.uk
Received January 3, 2005; Accepted February 17, 2005
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ABSTRACT
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In this review, we look at the most recent studies of DNA elements
that function over long genomic distances to regulate gene transcription
and will discuss the mechanisms genes employ to overcome the
positive and negative influences of their genomic neighbourhood
in order to achieve accurate programmes of expression. Enhancer
elements activate high levels of transcription of linked genes
from distal locations. Recent technological advances have demonstrated
chromatin loop interactions between enhancers and their target
promoters. Moreover, there is increasing evidence that these
dynamic interactions regulate the repositioning of genes to
foci of active transcription within the nucleus. Enhancers have
the potential to activate a number of neighbouring genes over
a large chromosomal region, hence, their action must be restricted
in order to prevent activation of non-target genes. This is
achieved by specialized DNA sequences, termed enhancer blockers
(or insulators), that interfere with an enhancer's ability to
communicate with a target promoter when positioned between the
two. Here, we summarize current models of enhancer blocking
activity and discuss recent findings of how it can be dynamically
regulated. It has become clear that enhancer blocking elements
should not be considered only as structural elements on the
periphery of gene loci, but as regulatory elements that are
crucial to the outcome of gene expression. The transcription
potential of a gene can also be susceptible to heterochromatic
silencing originating from its chromatin environment. Insulator
elements can act as barriers to the spread of heterochromatin.
We discuss recent evidence supporting a number of non-exclusive
mechanisms of barrier action, which mostly describe the modulation
of chromatin structure or modification.
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INTRODUCTION
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Metazoan locus control regions (LCR) and enhancer elements activate
high levels of transcription of linked genes from distal locations.
Although most enhancers are located tens of kilobases away,
some have been found at distances of up to a megabase from their
gene target (1
–3
). Enhancer elements, therefore, have
the potential to activate a number of neighbouring genes over
a large chromosomal region. Although some enhancers in fruit
flies display some preference towards core promoter or promoter
proximal elements, most enhancers appear to be promiscuous (4
,5
).
Hence, the action of enhancers must be restricted in order to
prevent the activation of non-target genes within their long
reach. The transcription potential of a gene is also susceptible
to silencing from its chromatin environment. A major fraction
of vertebrate genomes is composed of gene-poor repetitive DNA
and inactive genes that can exist as extended regions of condensed
chromatin (6
,7
). Chromatin condensation processes are self-propagating
and can spread into neighbouring gene loci, potentially affecting
their expression (8
). This is often observed when randomly integrated
genes become subjected to chromosomal position effect silencing
in transgenic cells or animals. Chromosomal position effect
silencing has also been observed following translocations that
result in human genetic disease (9
,10
). Genes must, therefore,
employ mechanisms to overcome the positive and negative influences
of their genomic neighbourhood, if they are to achieve accurate
temporal, spatial and responsive modes of expression.
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DEFINING CHROMATIN BOUNDARIES
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It has been proposed that genes and gene clusters may achieve
regulatory autonomy by organization into chromatin domains that
are maintained independently of their surroundings through the
establishment of chromatin boundaries (reviewed in 11
). Chromatin
boundaries are observed as a physical transition from transcriptionally
repressive condensed chromatin to more permissive open chromatin
structures. Boundaries can be the result of a balance between
countervailing chromatin opening and condensation processes
that are nucleated at neighbouring gene loci. Such boundaries
would not be fixed, so large intergenic regions would be required
to buffer a gene from its neighbours. Alternatively, specialized
DNA sequences, termed insulators, can establish boundaries of
fixed location (11
). Insulator elements found from yeast to
man share a common ability to protect genes from inappropriate
regulatory influences from their neighbours and are functionally
defined as having either or both of two activities in reporter
assays. An element with enhancer blocking activity interferes
with the communication between a linked enhancer and promoter
when positioned between the two, but it has no or little effect
when positioned at either side. In contrast, an element with
heterochromatin barrier activity halts the propagation of transcriptionally
repressive condensed chromatin structures only when positioned
between sequences that nucleate heterochromatin and the gene
promoter vulnerable to silencing.
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DEFINING LCRs
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Examination of ß-globin gene sequences in Dutch
ß-thalassemia
patients in the early 1980s revealed their gene structure to
be normal but found that a large deletion of upstream sequences
was associated with abnormal ß-globin gene expression
and disease (12
). Subsequent work showed that the deletion removed
a number of DNase I hypersensitive sites (DHSs) (13
) in a regulatory
region now known as the ß-globin LCR (14
). LCRs were
functionally defined in transgenic mice through characterization
of the human ß-globin and
CD2 LCRs (14
,15
). Since
then dozens of LCRs have been described for various genes (reviewed
in (16
). The property that set LCRs apart from other regulatory
elements was their ability to drive expression of a linked transgene
at a level equivalent to its endogenous counterpart from any
position in the genome (14
,17
,18
). Thus, LCRs were hailed as
the sequences both necessary and sufficient for full, position-independent
expression of a transgene, suggesting a role in opening up extensive
chromatin domains. Considering this broad definition, it is
understandable that many genomic regions defined as LCRs have
since been found to contain or to be associated with a variety
of regulatory elements, often combining classical enhancers
and insulators (19
–21
). Although the feature of position-independent
transgene expression with LCRs is well established, the suggestion
that LCRs controlled widespread chromatin opening was contradicted
by LCR knockouts (22
,23
) and chromatin structural studies showing
that LCRs do not necessarily control the chromatin environment
of their target genes (24
). Recent results imply LCR-like elements
have a primary role in transcription enhancement of poised or
epigenetically potentiated genes that need not be related in
structure or function (24
–26
). For example, the human
B-cell-specific gene
Igß is located between the pituitary-specific
human growth hormone (
hGH) gene and its LCR. High levels of
seemingly non-functional
Igß transcripts are detected
in the pituitary of humans and transgenic mice carrying the
hGH locus, suggesting that genes can be activated as innocent
bystanders if positioned within an active chromatin domain (25
).
Remote control by long-range elements led to new perspectives in the genetic analysis of specific diseases in which the suspected target genes appeared to be completely normal. For example, coding regions mutations in the SOX9 gene have been shown to be responsible for autosomal sex reversal and campomelic dysplasia (CD); however, many cases of CD were found in which the SOX9 gene was not disrupted but associated with distal genomic lesions (27,28). Transgenic experiments support the analysis of translocation breakpoints and suggest that several SOX9 regulatory elements are located hundreds of kilobases upstream of the gene (29,30). Similarly, disruption of long-range regulatory elements up to a megabase from the sonic hedgehog (Shh) gene have been implicated in Shh misexpression and resultant preaxial polydactyly (1,31). The fact that regulation can be achieved from considerable genomic distances has engendered lively debate on the mechanisms of long-range gene control. Technical advancements in recent years have permitted a breakthrough in elucidating the contribution of LCRs and other genomic elements to the higher-order folding of chromatin domains through long-range chromatin interactions.
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BRIDGING THE GAP
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The models of long-range enhancer action fall into two basic
categories: those that involve contact via direct interaction
with the target gene and those that propose no contact. Two
recently developed techniques: capturing chromosome conformation
(3C) (32
,33
) and tagging and recovery of associated proteins
(RNA TRAP) (34
) have provided compelling evidence in favour
of the former hypothesis. The 3C assay involves formaldehyde
crosslinking of neighbouring chromatin regions in cells, followed
by restriction enzyme digestion. After dilution to very low
DNA concentration, intermolecular crosslinked chromatin fragments
are ligated and detected via PCR across the novel ligation junctions.
Several recent reports detail the use of this technique in the
analysis of higher-order folding of specific loci and two groups
have used 3C to investigate LCR–gene interactions in the
ß-globin and T
H2 cytokine loci (32
,35
–38
).
The RNA TRAP technique directs horseradish peroxidase (HRP) activity to the site of a transcribing gene via a modified RNA FISH procedure. The localized HRP activity then catalyses the activation and subsequent covalent attachment of a biotinyl-tyramide tag onto chromatin proteins in the immediate vicinity of the transcribing gene. Tagged chromatin components are then recovered and the associated DNA analysed by PCR. RNA TRAP analysis of the transcribing mouse ß-globin genes found enrichment of specific DHSs of the ß-globin LCR (50 kb upstream) implying close association with the transcribing gene, whereas inactive intervening genes and distal upstream and downstream DHSs were not enriched (34). Similar interactions between the active gene and LCR were inferred from 3C assays but ligation products for many other DHS elements both upstream of the LCR and downstream of the ß-globin genes were also detected, implying that many elements over a 150 kb region participate in the formation of what has been called an ‘active chromatin hub’ (ACH) (37). The additional upstream and downstream DHS elements detected by 3C appeared to associate prior to, or in the absence of, active gene transcription (36,38), suggesting that an initial chromatin hub is formed between DHSs not directly involved in transcriptional activation of the genes. Interestingly, many of the regions that associate in the chromatin hub are capable of binding the protein CTCF and have enhancer-blocking activity (19,20) (discussed subsequently). Evidence for the formation of a precursor higher-order structure was also found in the TH2 cytokine locus (35). In this case, genomic elements in the gene promoter regions formed an initial chromatin core configuration that was evident in non-expressing cell types and naive T-cells prior to transcriptional activation of the cytokine genes. Inclusion of the LCR in the ACH complex was found in differentiated cell types that expressed the cytokine genes.
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ORGANIZING THE HUB FOR TRANSCRIPTION
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What is missing from studies detailing interactions among LCRs,
genes and other genetic elements is the key that explains how
higher-order structure activates high-level gene transcription.
It has been known for some time that gene positions in the nucleus
often reflect activity states (39
–43
). In some cases,
silenced genes have been found to associate with centromeric
heterochromatin or the nuclear periphery, whereas active genes
were found in more internal positions in the nucleus. Gene activity
has also been linked to position relative to its chromosome's
territory. In the mouse
HoxB cluster, the sequential activation
of genes was related to chromatin decondensation and nuclear
reorganization (44
). Concurrent with activation, an early expressed
gene appeared to loop out of its chromosome territory and away
from other late expressed genes in the cluster, only to be followed
at the appropriate stage by a late expressing gene. Although
extra-territorial positioning has been correlated with the transcriptional
activity of several genes, transcription can also occur inside
what is loosely defined as the chromosome territory (45
). What
has become increasingly clear is that individual genes are moving
in association with function (46
). Exactly where active genes
are going was addressed in a recent study, showing that potentiated
genes migrate to nuclear foci enriched in RNAP II and transcriptional
machinery, known as transcription factories (Fig.
1) (47
).
Interestingly, many active genes over a 40 Mb region of
mouse chromosome 7 ended up in the same transcription factory
as the LCR-driven ß-globin genes, indicating that
genes migrate to pre-assembled, shared sites of transcription.
The available evidence suggests that transcription factories
are at least meta-stable structures (48
,49
), possibly formed
through the principles of self-organization whereby the structure
is formed and stabilized by the addition of interacting components
and transcription units (50
).
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Figure 1. Long-range chromatin interactions upon gene activation. Recent studies have shown that upon their transcriptional activation, genes can migrate to the edge of, or beyond, their chromosomal territories (grey) to foci enriched in RNA polymerase II and transcriptional machinery, known as transcription factories (red disk). Gene promoters (arrows) have been observed to be in close physical proximity to linked enhancer elements (circles) upon transcription. Trans-acting factors (diamonds) recruited to enhancers and promoters determine interactions between looped chromatin fibres (gold). Different chromatin loops may form depending on the tissue-specific expression of trans-acting factors. Overlapping tissue-specific gene loci may operate independently from one another owing to the absence of their respective regulatory factors in one another's tissue. Alternatively, overlapping gene loci may share common enhancers of broad function. Promoter specificity factors or enhancer-blocking elements will be required to determine the correct promoter choice and loop interactions in such a situation.
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FACILITATING ENHANCER–PROMOTER CONTACT
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The ease of looping between a gene and distal enhancer is determined
by the intrinsic flexibility of the chromatin fibre. By modelling
the chromatin fibre as a polymer, it has been calculated that
the three-dimensional juxtaposition of elements separated by
several kilobases would be inefficient (51
). This is supported
by the finding that site-specific recombination between elements
separated by several kilobases is inefficient in mammalian cells
compared with interactions within a kilobase (52
). It appears
unlikely that unassisted looping would be either efficient or
specific. Most importantly, it is hard to reconcile the position
dependence of enhancer-blocking elements with a free diffusion
model of enhancer looping. Rather, the position dependence of
enhancer-blocking elements indicates that the initial communication
between an enhancer and promoter is directly linked to the intervening
chromatin fibre. We suggest that the following order of events
leading to enhancer–promoter contact (Fig.
2). Step
1: LCR/enhancer-bound factors direct the assembly of, or migration
to, a transcription factory. Step 2: the enhancer reels in the
chromatin fibre in
cis in search of a potentiated promoter.
Step 3: interactions between enhancer- and promoter-bound factors
stabilize the association of the promoter with the transcription
factory. This model takes into account the
cis positional dependence
of enhancer-blocking elements in the context of looping interactions
between genomic elements. It is quite conceivable that enhancer
contact with the high concentration of immobilized RNA polymerase
at a factory (49
,53
) in step 1 could lead to the initiation
of intergenic transcription. The powerful motor protein activity
of RNA polymerase (54
), and associated chromatin remodelling
enzymes (55
), would facilitate reeling of the chromatin fibre
in step 2. Enhancer-blocking elements could interfere with step
2 (56
,57
) by preventing gene access to a factory (Fig.
2D).
In this scenario, it is likely that nuclear relocation (44
)
and the initial engagement of regulatory elements or their target
genes with transcription factories (47
) are critical steps that
determine expression levels, especially in light of the finding
that most ‘active’ genes are not continually transcribed,
but appear to move in and out of factories with corresponding
oscillations in transcription (47
).
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Figure 2. Events that might lead to enhancer-promoter contact. (A) Step 1: LCR/enhancer-bound factors direct the assembly of, or migration to, a transcription factory. (B) Step 2: The chromatin fibre is reeled in (black arrows) to search for a potentiated promoter (red) in cis. (C) Step 3: Interactions between enhancer- and promoter-bound factors stabilize the association of the potentiated promoter with the transcription factory to permit high levels of gene transcription (red arrow). (D) An enhancer-blocking element (blue) could interfere with chromatin fibre repositioning in step 2 in order to block a gene's access to a factory. Refer to text for models of enhancer blocking.
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An alternate, though not necessarily exclusive, view is based
on the finding that a number of highly conserved transcription
factors have been described to facilitate long-range enhancer–promoter
communication (58
). For example, the conserved LIM-domain binding
protein chip, which is required for maximal activation by a
diverse set of enhancers in
Drosophila, is proposed to act as
a protein crosslinker. Chip can form cooperative interactions
between LIM proteins nucleated at an enhancer and homeodomain
proteins, whose binding sites are frequently interspersed between
enhancers and promoters. Chip-assisted homeodomain binding may
occur in a processive manner along the chromatin fibre, eventually
bringing enhancer and promoter complexes into close proximity
(59
). Another enhancer facilitator protein, Nipped-B, is related
to the adherins, which promote the loading and removal of cohesin
(60
). Remodelling of cohesin anchors may facilitate the movement
of a chromatin fibre beyond its chromosomal territory.
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SETTING THE BOUNDARIES OF ENHANCER ACTION
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Although a number of enhancer-blocking proteins have been identified
in
Drosophila (11
), CTCF so far exists as the only protein known
to mediate enhancer-blocking activity in vertebrates. CTCF sites
have been found at vertebrate enhancer blocking elements that
function in a wide variety of contexts (20
,61
–74
). Evidence
from several systems has led to the proposal of a number of
mechanisms to explain enhancer-blocking activity (Fig.
3).
Enhancer blockers may mimic the characteristics of a promoter
that mediate interactions with an enhancer. For example, enhancer-blocking
proteins can interact with facilitator proteins to terminate
enhancer-promoter communication (Fig.
3A) (75
). The problem
with this model is that a so-called decoy element placed outside
a linked enhancer and promoter could sequester the enhancer
and render it unproductive, thus behaving as a bidirectional
silencer rather than a position-dependent enhancer blocker.
However, position dependence may still be achievable if enhancer–decoy
interactions are transient. If this is the case, a decoy element
placed outside a linked enhancer and promoter may only reduce
enhancer activity by a small fraction, but would repeatedly
block enhancer activity when positioned between an enhancer
and its target.
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Figure 3. Potential mechanisms of enhancer-blocking. (A) Mimic or decoy: enhancer blockers (blue) may mimic the characteristics of a promoter that mediates interactions with enhancer factors (red). For example, enhancer-blocking proteins can interact with facilitator proteins (green) to terminate enhancer–promoter communication. (B) Distraction: an enhancer may be ‘mis-directed’ (red arrows) by the close proximity of other chromatin fibres (gold) at the base of loops tethered by enhancer blockers, resulting in a dilution of the enhancer's efforts to communicate with a particular target gene (green). (C) Obstruction: an enhancer may be blocked from making interactions with promoters beyond their own loop by steric interference at the base of the loop.
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Another model for enhancer blocking involves the tethering to
other elements or fixed structures in the nucleus. The
gypsy element from
Drosophila is observed to be tethered to foci near
the nuclear periphery, whereas the chicken HS4 element has been
found to be tethered to the nucleolar periphery in human cells
(76
–78
). It appears unlikely that the nuclear localization
resulting from tethering to a fixed structure would determine
enhancer-blocking activity, as any affects of localization on
enhancer activity would not be position dependent. Consistent
with this view, it has been shown that different sequences within
the
gypsy element mediate enhancer blocking and localization
towards the nuclear periphery (78
). Alternative interpretations
are based on the observation that the tethering of multiple
enhancer-blocking elements to the same foci can lead to the
formation of chromatin loops (79
). By using DNA FISH on nuclear
halo preparations, probes located between two endogenous
Drosophila insulators revealed that the intervening chromatin is arranged
in a loop, with the two insulators located at the base. Furthermore,
insulators were shown to cause the chromatin looping. Insertion
of an additional insulator in the centre of the loop results
in the formation of two smaller loops, and mutations of insulator-binding
proteins lead to the disruption of the loop (79
). Tethering
of an enhancer blocker to foci could interfere with an enhancer's
access to a gene promoter in two ways. First, an enhancer may
be ‘mis-directed’ by the close proximity of other
chromatin fibres at the base of the tethered loops, resulting
in a dilution of the enhancer's efforts to communicate with
the target gene (Fig.
3B) (11
). There is evidence of such
trans enhancer action in
Drosophila, where homologous chromosomes
are paired somatically (80
). Secondly, enhancers may be ‘obstructed’
from making interactions with promoters beyond their own loop
by torsional constraints or steric interference at the base
of the loop (Fig.
3C). In this scenario, the choice of
which neighbouring elements an enhancer blocker pairs with would
be critical in determining which loop an enhancer is restricted
to (reviewed in 81
). This model would also depend on enhancer–promoter
communication being linked to the chromatin fibre, otherwise
there is little to stop enhancer–promoter communication
between loops. For example, chromatin reeling driven by intergenic
transcription from an enhancer might be obstructed at the base
of the tethered loops. In support of this model, it has recently
been demonstrated that the blocking of intergenic transcription
from a ß-globin enhancer can interfere with its long-range
enhancement in a transgenic assay (56
,57
).
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REGULATING ENHANCER BLOCKING
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Recent studies have revealed a number of means by which enhancer-blocking
elements can be regulated (Fig.
4). The binding of the
vertebrate enhancer-blocking protein CTCF can be abrogated by
CpG methylation, for example. The establishment of differential
DNA methylation patterns can, therefore, be employed as means
to regulate enhancer access, as observed at many imprinted gene
loci (61
,62
,64
,66
,67
,73
,74
). The regulation of CTCF binding
in this manner means that enhancer-blocking elements are sensitive
to aberrant DNA methylation observed during disease progression,
potentially resulting in deleterious enhancer crosstalk (63
,82
–85
).
It was recently shown that CTCF interacts with poly(ADP-ribose)
polymerase (PARP) and that it is itself poly(ADP-ribosyl)ated
(76
,86
). Poly(ADP-ribosyl)ation is associated with a number
of enhancer-blocking elements, whose activity is dependent on
PARP activity (86
). Without studying modification site mutants
of CTCF, it is unclear whether it is the modification of CTCF
itself, or some other factor, that is required for enhancer-blocking
activity of these elements. For example, the poly(ADP-ribosyl)ation
of histones, associated with chromatin opening (87
), may play
an essential role in enhancer blocking at CTCF elements. Regardless,
this finding highlights how the regulation of this post-translational
modification may allow genome-wide control of insulators which
may be critical during the setting of imprinted marks in germlines,
for example.
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Figure 4. Regulating enhancer blocking activity. (A) Enhancer blocking mediated by enhancer-blocking proteins (blue). Recent evidence has shown that enhancer blocking can by regulated by a number of means. (B) The binding of enhancer-blocking proteins can be abrogated by DNA methylation (black lollipops) of their binding sites. (C) Post-translational modification of enhancer-blocking proteins (yellow stars) may be required to facilitate their activity, potentially by regulating their interactions with other proteins. Alternatively, modifications (green stars) may abrogate the activity of enhancer-blocking proteins, potentially by obstructing their interactions with proteins or their DNA sites. (D) Enhancer blocking has also been shown to be regulated by protein co-factors (purple) that may regulate the interactions of enhancer-blocking proteins.
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Enhancer blocking has also been shown to be modulated by a number
of protein co-factors. In vertebrates, the activity of CTCF-binding
elements can be modulated by neighbouring binding sites for
thyroid hormone receptor, TR (65
). The enhancer-blocking activity
of composite CTCF/TR binding elements, which are found at several
gene loci, is abrogated in a hormone-dependent manner. This
form of regulation can allow otherwise constitutive enhancer-blocking
elements to be modulated in specific tissues during development
and differentiation.
Transgenic assays in Drosophila point to a third way in which protein co-factors can lead to the regulation of an enhancer blocker, termed ‘bypass’. The first evidence of bypass came from experiments where two copies of the gypsy insulator positioned in tandem neutralize one another's enhancer-blocking activity (88,89). It is postulated that closely spaced gypsy insulators physically pair with one another. We suggest that pairing interactions disrupt the enhancer-blocking activity of this insulator by interfering with its ability to form or tether to nuclear foci, although this remains to be tested. The enhancer-blocking activity of the gypsy element is dependent on the DNA-binding protein Su(Hw) and its interaction partners mod(mdg4) and CP190 (11,90). Mod(mdg4) and CP190 mediate protein–protein interactions via their BTB domains to direct tethering to nuclear foci, and presumably mediate self-pairing. It was recently demonstrated that natural elements containing arrays of binding sites for the BTB protein GAGA protein can also allow an enhancer to bypass the gypsy enhancer-blocking element (91). This bypass of gypsy is dependent on the presence of both GAGA and mod(mdg4), which can efficiently interact with one another through their BTB domains. Pairing may be a common means of mediating bypass of the subgroup of enhancer-blocking elements that function by tethering to nuclear foci. Dynamic regulation of enhancer blocking is achievable by regulating the expression of proteins that interact with the pairing elements, such as the BTB factors, which can then in turn interfere with adjacent enhancer blockers.
It remains to be demonstrated whether pairing interactions are used to regulate the activity of enhancer blockers in their natural contexts. We suggest that the anti-insulator component of the promoter targeting sequence (PTS), which is located adjacent to the Fab-8 insulator of the Drosophila Abd-B homeotic gene locus, may function as a pairing module (92). The PTS element can abrogate the enhancer-blocking activity of both the Fab-8 and the gypsy insulators to permit enhancer bypass in specific-cell types (92,93). It is tempting to speculate that the PTS may pair with Fab-8 in a cell-type specific manner. It should be cautioned that not all elements that function through BTB factors can mediate bypass of the gypsy element (94). Furthermore, other well characterized Drosophila enhancer blockers that do not use BTB factors to mediate their interactions are not sensitive to tandem pairing (94–96). Limited evidence suggests that CTCF-mediated enhancer blocking in vertebrates is not sensitive to tandem pairing. For example, two tandem copies of the chicken ß-globin HS4 element display normal enhancer blocking in cell culture or when inserted into mice to substitute for the Igf2/H19 ICR element (97,98). Furthermore, a duplicated Igf2/H19 ICR element also retains its CTCF-mediated enhancer blocking function (99). Nevertheless, the principle of bypass of enhancer blocking has been established and may occur at vertebrate gene loci with complex patterns of regulation.
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DEFINING THE BOUNDARIES OF CHROMATIN STATES
|
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Insulator elements were first identified at defined boundaries
between open and condensed chromatin domains (11
). These boundary
elements were found to possess both enhancer blocking and heterochromatin
barrier activities, but recent dissection of insulators has
revealed that these activities are separable. For example, the
enhancer-blocking activity of the chicken ß-globin
HS4 element is dependent on a single binding site for the protein
CTCF, whereas binding sites for other proteins including USF
are required for its barrier activity (97
,100
,101
). The
Drosophila SF1 insulator also has separable enhancer blocking and barrier
activities (102
) (H. Cai, personal communication). Furthermore,
CTCF-binding enhancer-blocking elements from several diverse
gene loci do not harbour barrier activity (101
). The barrier
activity of the HS4 element has proved useful in protecting
transgenes from chromosomal position effect silencing in a wide
variety of contexts in vertebrates (103
). It is necessary to
flank transgenes with barrier elements to prevent silencing,
suggesting that they interfere with the spread of silencing,
rather than promote activation. Indeed, the removal of barrier
elements or their binding proteins leads to the spread of heterochromatin
markers beyond their natural boundaries (100
,104
). Heterochromatin
from yeast to man is understood to assemble in a stepwise process,
originating at a nucleation site from which it can spread onto
nearby sequences (reviewed in 8
). For example, the methylation
of H3-lysine 9 creates a binding site for chromo-domain adaptor
proteins such as heterochromatin protein 1 (HP1). HP1, in turn,
can recruit SUV39H1, an H3-lysine 9-specific histone methyltransferase,
in principle allowing further methylation of H3-lysine 9 and
HP1 binding to extend onto successive nucleosomes in a self-propagating
fashion. Equivalent mechanisms for the propagation of silencing
may involve other modifications and their associated proteins,
such as histone deacetylation and Sir proteins in yeast or the
methylation of H3-lysine 27 and the polycomb group of proteins
in metazoa.
Studies of barrier elements from yeast to vertebrates have led to the development of several models of barrier activity (Fig. 5). One passive mechanism is the tethering of barrier elements to fixed structures, which may create a steric hindrance to propagation mechanisms. It has been shown that artificial tethering to the nuclear pore complex (NPC) can form an efficient barrier to silencing at the HML locus in budding yeast, for example (Fig. 5A) (105). Many yeast genes localize to nuclear pores upon their transcriptional activation indicating that this may be a compartment of the nucleus particularly favourable to transcription (39). Vertebrate genes, on the other hand, show no preferential association with nuclear pores when transcribed, although a comprehensive screen has not been performed. However, targeting to a compartment does not explain the position dependence of barrier elements or the necessity to flank a reporter gene with NPC barriers. Furthermore, although tethering to the NPC can form a barrier to silencing at the HML locus, it does not block silencing at HMR (106). It is possible that distinct modes of silencing exist at each of these loci, with an NPC-tethered barrier having a different level of effectiveness against each. Tethering to fixed structures has not been demonstrated at natural barriers to date. Interestingly, a mutation of the CTCF-binding site within the chicken HS4 insulator element abrogates its tethering and enhancer-blocking activity but has no affect on its barrier activity (76,101).
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Figure 5. Potential mechanisms of barrier action. (A) Tethering: it was recently shown that artificial tethering to a fixed structure such as the NPC (black) can form an efficient barrier (purple) to the spread of histone modifications (blue dots) and heterochromatin factors (blue) associated with repression. Flanking with barriers (purple) can help a transgene to establish an active chromatin domain (green). (B) Nucleosome gap: multiple binding sites for some barrier proteins (purple) have been shown to exclude nucleosomes, thereby interrupting the substrate required for further heterochromatin factor binding. (C) Nucleosome masking: it was recently shown that a barrier protein can compete with heterochromatin propagation by binding directly to histones. (D) Histone code manipulation: evidence from several natural barrier elements has revealed that barrier proteins often recruit multiple histone modifying enzymes (green). Histone modifications associated with activation (green dots) in the immediate vicinity of the barrier can compete with the propagation of heterochromatin factor binding.
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Another passive mechanism involves the creation of a nucleosome
gap, thereby interrupting the substrate that heterochromatin
factors require (Fig.
5B) (107
). This can be achieved through
multiple sites for DNA-binding proteins that prevent nucleosome
placement. A variation on this model is nucleosome masking,
where barrier-binding proteins also interact with histones (Fig.
5C).
For example, the NFI transcription factor family protein CTF-1,
whose binding sites can form a barrier to silencing at budding
yeast telomeres, specifically interacts with the histone variant
H3.3 (108
). It is proposed that CTF-1 competes with the binding
of co-repressor proteins such as the histone deacetylase Sir2
to block the propagation of heterochromatin. A more substantial
and active approach to barrier formation involves manipulation
of the histone code (Fig.
5D). The modification of histones
is a common component of native chromatin boundaries in budding
yeast (109
). Furthermore, it has been demonstrated that the
artificial tethering of histone acetyltransferases to chromatin
is sufficient to form a barrier to heterochromatin in yeast
(110
). The barrier activity of the most well characterized vertebrate
insulator element, chicken HS4, is also dependent on a number
of histone modifications. Nucleosomes in the immediate vicinity
of the HS4 element are acetylated at multiple sites on H3 and
H4 and methylated at H3-lysine 4 (111
,112
). It was recently
found that the ubiquitous transcription factor USF is responsible
for recruiting a number of histone modifying enzymes responsible
for the modification profile of the HS4 element (100
). These
findings have lead to a chain terminator model of barrier activity,
where activating histone modifications localized at a barrier
element interrupt the propagation of silencing histone modifications
by chromatin condensation complexes. Consistent with this view,
removal of the USF binding site or knock down of USF expression
results in a loss of HS4 barrier activity, leading to an encroachment
of silent chromatin marks over the barrier (100
). Active histone
modifications may contribute to barrier activity either by blocking
the modification of the same histone residue required for silencing
or by preventing the interaction of co-repressor complexes with
histones. In striking similarity to HS4, peaks of H3-lysine
4 methylation also flank the active chromatin of the poised
DJ regions of the murine
IgH and
TCRß antigen receptor
loci (113
). It remains to be seen whether these regions also
have barrier activity, but constitutive peaks of H3-lysine 4
methylation between gene loci may be a hallmark of barrier elements
in vertebrates. Although the recruitment of active histone modifications
is a necessary component of the HS4 barrier, it is not sufficient
(100
). This marks a clear distinction between this vertebrate
barrier and those characterized in yeast. Mutagenesis of HS4
has found that three further protein binding sites, which do
not contribute to the studied histone modifications at HS4,
are also required for barrier activity (101
). It is clear that
this compound element still has stories to tell.
|
OUTSTANDING QUESTIONS
|
|---|
Recent technological advances have rekindled investigations
into the long-range control of gene transcription. It has been
demonstrated that enhancers are in close physical proximity
to their target gene promoters in two out of two gene loci studied
so far. It remains to be seen whether all enhancers mediate
promoter contact, especially those that are located at distances
of up to a megabase from their target promoters. Although the
3C and related methods will allow the study of enhancer–promoter
contact at other well characterized gene loci, we suggest that
it might also be possible to use the 3C approach to trap novel
regulatory elements that are in physical proximity with a known
gene promoter of interest. We have proposed a sequential model
of the events that might lead to enhancer–promoter contact.
It remains to be determined whether enhancers locate to, or
nucleate, transcription foci prior to gene transcription. It
may now be possible to study this at extended mammalian gene
loci whose genes undergo considerable nuclear repositioning
upon their transcription. It would be of great interest to observe
what happens to their nuclear repositioning when an enhancer
blocker is inserted between a distal enhancer and promoter.
Will the enhancer blocker prevent repositioning of the gene?
Will the enhancer blocker be in contact with the enhancer?
While insulator elements and their properties have been known for some time, we are just beginning to identify vertebrate insulators and understand their contributions to gene expression. We do not know how common insulators are in the human genome, how many have enhancer-blocking activity or how many have barrier activity. The recent identification of the first conserved vertebrate insulator proteins from studies of the chicken HS4 element now allows protein-led screens for novel elements. Only when we have detailed information from a large set of insulators can we assess their overall contribution to regulating transcription of the genome. The identification of insulator proteins also now allows a detailed investigation of their mechanism of action. More biochemistry is required to reveal their binding partners and/or the enzymatic activities associated with insulators. The most well studied vertebrate insulator HS4 has served as a paradigm constitutive element with both enhancer-blocking and barrier activities. Yet, since the identification of CTCF, none of the newly identified enhancer-blocking elements has been shown to have barrier activity. Are barrier elements uncommon or are they just rarely co-located with enhancer-blocking elements? In addition, a number of the human enhancer-blocking elements found to date are regulated. Can barrier elements also be regulated? Considerable progress has recently been made in beginning to identify and understand the elements we have discussed here. We now have the tools to address fundamental questions regarding their mechanisms and their roles in the remote control of gene transcription.
|
ACKNOWLEDGEMENTS
|
|---|
We are grateful to our colleagues, particularly Miklos Gaszner,
Katherine West and the guest editors for their helpful discussions.
Our laboratories are supported by the UK Biotechnology and Biological
Sciences Research Council and Medical Research Council (MRC)
and by the European Commission.
|
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TOP
ABSTRACT
INTRODUCTION