Download Facilitation of chromatin dynamics by SARs Craig M Hart and Ulrich

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Facilitation of chromatin dynamics by SARs
Craig M Hart and Ulrich K Laemmli*
Metaphase chromosome condensation is a dynamic process
that must utilize cis elements to form and maintain the final
structure. Likewise, cis elements must regulate the
accessibility of chromatin domains to protein machines
involved in processes such as transcription. Scaffold
associated regions appear to play important roles in both of
these dynamic processes.
Addresses
Departments of Biochemistry and Molecular Biology, University of
Geneva 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland
*e-mail: [email protected]
Current Opinion in Genetics & Development 1998, 8:519–525
http://biomednet.com/elecref/0959437X00800519
(A tracts), the non-B DNA structure (narrow minor
groove, bends, propensity to unwind) of which are recognized by certain SAR-binding proteins such as histone H1
and topoisomerase II (reviewed in [4]). It has been difficult to address and decipher the in vivo role of SARs
because of their non-standard sequence determinants and
the absence of a convenient assay system that allows functional studies. Despite these difficulties, the recent
observations reviewed here show that progress is being
made in understanding SAR function. Structural studies
demonstrate that SARs are not arranged randomly but
appear juxtaposed in metaphase chromosomes as is
expected from the loop-scaffolding model, whereas other
studies strongly implicate SARs as genuine cis elements of
chromosome dynamics and gene expression.
© Current Biology Ltd ISSN 0959-437X
Abbreviations
PEV
position effect variegation
SARs
scaffold associated regions
Introduction
Various structural studies have demonstrated that the chromosomal architecture of the nucleus is highly ordered and
suggest that it might be held in place by some sort of
nuclear scaffold. This organization must permit dynamic
movements of chromatin during activities such as
enhancer looping, recombination and chromosome condensation. The mechanisms whereby nuclear order and
chromatin dynamics are achieved remain enigmatic.
These biological processes are presumably governed by cis
elements that are distributed at intervals along the
genome. Activities mediated by such cis elements presumably include tethering chromatin to some kind of nuclear
scaffold, serving as signalling or initiator stations from
which open, closed or heterochromatic chromatin states
spread, and limiting the spreading of these chromatin
states. Different types of cis elements could be involved in
these activities, although it is also possible that one type of
cis element could govern more than one type of activity.
Scaffold associated regions (SARs; also called matrix
attachment regions [MARs]) are the only candidate cis elements identified to date that are implicated in facilitating
dynamic changes in chromatin states and in the structural
organization of chromosomes.
SARs are very AT-rich fragments several hundred base
pairs in length that were first identified as DNA fragments that are retained by nuclear scaffold/matrix
preparations [1,2]. They define the bases of the DNA
loops that become visible as a halo around extracted
nuclei and that can be traced in suitable electron micrographs of histone-depleted metaphase chromosomes [3].
They are possibly best described as being composed of
numerous clustered, irregularly spaced runs of As and Ts
SARs and metaphase chromosome structure
Numerous structural studies support the view that the
chromatin fiber of metaphase chromosomes is organized
into loops (reviewed in [5]). If SARs define the bases of
the loops in mitotic chromatin and are juxtaposed by the
scaffolding, then native chromosomes should have an ATrich subregion where the SARs ‘queue up’. Using special
staining techniques combined with optical sectioning by
confocal microscopy and image reconstruction [6], this ATqueue was visualized in the giant chromosome of the
Indian muntjac (Figure 1a,b). The AT-queue proceeds
through the chromosomal cylinder on an irregular, helicallike path that is stretched longitudinally at certain
intervals (Figure 1c).
Remarkably, these structural studies suggest that classic
chromosome banding patterns (reviewed in [7]) are manifested by the differential folding pattern of the AT-queue
rather than by a large difference in the base composition
between Q and R bands (Figure 1c). The Q-bands (also
known as G-bands) obtained with classic staining methods appear morphologically ‘AT-rich’ as they are in
regions where the AT-queue is more tightly coiled/folded.
In contrast, R bands appear morphologically ‘AT-poor’ as
they are in regions where the AT-queue is more unfolded
and centrally positioned (Figure 1c). R-bands are known
to replicate early, to contain most housekeeping genes
and are enriched in hyperacetylated histone H4 [8] and
DNase I-sensitive chromatin [9]. This suggests they have
a more open chromatin conformation, consistent with a
central AT-queue with longer loops that reach the nuclear
periphery. In contrast, Q-bands contain fewer genes and
are proposed to have loops that are shorter and more tightly folded, resulting in an AT-queue path resembling a
coiled spring.
Whereas a more extended chromatin conformation is
thought to contribute to the longer loops of R-bands, a
520
Differentiation and gene regulation
Figure 1
(a) Chromatid : cross-section
Optical
sections
(~0.2 µm)
AT-queue:
juxtaposed SARs
~1.6 µm
(b) Loop : differential staining
SAR
Body
Daunomycin
YOYO/methyl green
~500nm (~100kb)
(c) Metaphase chromosome model
Scaffold
AT-queue
(SARs)
Q-Band
Q-Loops
R-Band
R-Loops
Q-Band
AT-coil
(Giemsa sub-band)
SARs appear juxtaposed along the scaffold of
metaphase chromosomes [6]. (a) Schematic
representation of a cross-section through a
chromatid. Chromosomal DNA is compacted
~10,000 fold during metaphase through a
hierarchy of folding principles. One of the final
levels of compaction is achieved by formation
of loops imposed by non-histone scaffold
proteins. If AT-rich SARs form the bases of
these loops and are juxtaposed by the
scaffold, then one would expect to find an ATrich subregion inside chromosomes (shown
as a light grey star) where SARs line up (the
AT-queue). Loops of non-SAR DNA would
emanate from this region (dark grey). To
visualize this, chromosomes thick enough to
allow optical sectioning by confocal
microscopy need to be used, such as those
from Indian muntjac cells. (b) Depiction of a
loop and the strategy used to detect SAR and
non-SAR sequences. The fluorescent
emission of daunomycin is highly enhanced by
AT-rich DNA such as SARs, whereas
quenching of the general DNA dye YOYO
with methyl green stains non-SAR DNA. (c)
Model of chromosome structure derived from
such experiments. Although chromatids are
fairly uniformly filled with DNA, an internal ATrich region can be discerned. This AT-queue
forms by scaffold-mediated juxtapositioning of
SARs. Classic chromosomal banding patterns
can be explained by the path of the AT-queue.
The AT-queue is relatively tightly folded (coillike) in gene-poor Q-band regions, and is less
folded and more central in gene-rich R-band
regions (see text for details). Loops are not
directly observed by fluorescence microscopy
but are inferred from the path of the AT-queue.
The structural relationships of neighboring
loops and their packaging modes are
unknown and are only schematically indicated.
Current Opinion in Genetics & Development
greater length of DNA between SARs may also contribute.
In support of this view, the 90 kb β-globin locus is in a
G-band and has 8 SARs mapped within it, whereas the
140 kb α-globin complex is in an R-band and no SARs are
found in this region [10]. Similarly, a study in which bulk
SAR and bulk non-SAR DNAs were used to paint chromosomes by in situ hybridization found that G-bands have a
higher ratio of SAR to non-SAR DNA than do R-bands [11•].
Topoisomerase II is a major protein of metaphase scaffolds
[12,13] that preferentially binds SAR DNA in vitro [14].
The structural studies using intact chromosomes discussed
here extend the notion that topoisomerase II follows the
scaffold as it was found that it and the AT-queue co-localize [6]. These observations support the view that the
scaffolding mediates chromatin-loop formation, presumably through interactions involving or facilitated by SARs.
SARs and chromosome dynamics
Are SARs involved directly in chromosome dynamics? The
role of SARs in chromosome condensation was tested by
using an artificial protein [15] that consists of numerous,
linked DNA-binding domains, called AT-hooks, which
preferentially interact with A tracts through minor groove
Facilitation of chromatin dynamics by SARs Hart and Laemmli
contacts [16]. MATH-20 is a multi AT-hook SAR-binding
protein composed of 20 reiterated AT hooks. As the
numerous A tracts of SARs can simultaneously accommodate all 20 hooks, this protein was found to bind SAR DNA
and SAR chromatin with both extremely high specificity
and affinity (see Figure 2a).
521
Assembly of metaphase chromosomes from added sperm or
somatic nuclei by Xenopus egg extracts is known to go
through a number of morphological intermediates to form
individual chromatids. A low dose of MATH-20, about one
molecule bound per 15 kb of DNA, was found to inhibit a
late stage of chromosome condensation. Under these con-
Figure 2
(a)
MATH: multi-AT-hook protein
AT-hook motif
20 hooks: MATH-20
Strong interaction
Weak interaction
DNA
SAR
Clustered A-tracts
(b)
Single A-tract
MATH-20 blocks chromosome assembly
Somatic nuclei
Chromatids
Chromatid balls
0 min
90 min
120 min
+ MATH-20
(90 min)
'Collapses'
the scaffold
Abortive mitotic structures
0 min
90 min
+ MATH-20
(0–10min)
SARs are necessary for mitotic chromosome assembly [15].
(a) Construction of a highly specific SAR-binding protein. MATH-20 is
a multi-AT-hook protein comprising of 20 reiterated AT hook peptide
motifs, derived from HMG-I/Y, linked with a spacer of ~25 amino
acids. As depicted, this protein is extremely specific for SAR DNA
and SAR chromatin, binding with high affinity because other
sequences cannot simultaneously accomodate all 20 hooks. (b)
MATH-20 blocks chromosome assembly. Xenopus egg extracts
Current Opinion in Genetics & Development
convert somatic nuclei (left) into metaphase chromatids (top center).
Titration of SARs with MATH-20 specifically inhibits this without
inhibiting condensation, resulting in abortive mitotic structures where
the condensed chromatin is located at the nuclear periphery
surrounding a near-empty center thought to arise from the
disassembly of nucleoli (bottom right). When added to formed
chromatids, MATH-20 interferes with their structural stability and they
collapse into individual spheres (‘chromatid balls’; top right).
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Differentiation and gene regulation
Figure 3
(a)
Immunoglobulin µ gene
Accessibility
T7
T3
SAR
Regional
demethylation
local
(T3)
distant
(T7)
+++
+++
+
+++
+/–
–
Enhancer
1 kb
(b)
+
W
m4
W
m4
W
+ MATH-20
Current Opinion in Genetics & Development
SARs affect gene regulation through chromatin-state transitions.
(a) Developmentally regulated opening of the immunoglobulin µ gene
utilizes a collaboration between an enhancer and associated SAR.
The µ gene has an intronic enhancer with an associated SAR. The
enhancer alone forms a DNase I hypersensitive site, but association
with a SAR is needed for developmentally regulated general
nuclease sensitivity and gene activation during B cell differentiation
[27]. In an elegant set of experiments [28 ••], it was further shown
that both accessibility of T7 RNA polymerase to a promoter located
1 kb from the enhancer and regional demethylation require the
enhancer and associated SAR (top), whereas the enhancer alone
only conferred local accessibility to the adjacent T3 RNA polymerase
promoter (bottom). The immunoglobulin κ-chain gene is similarly
regulated and also has an intronic enhancer with an associated SAR.
A similar study found that both the intronic SAR and associated
enhancer are necessary for the developmentally regulated
demethylation that precedes transcriptional activation of the κ-chain
gene [29,30]. The common cohabitation of SARs and enhancers
[31] suggests this could be a general mechanism for conferring
specificity to SAR-mediated chromatin state transitions. (b) MATH20 protein suppresses PEV caused by juxtaposition next to SAR-like
Satellite III sequences in Drosophila [32••]. The red pigmentation
(black here) of the eyes of wild-type flies requires the protein
encoded by the white gene (W+). A chromosomal inversion (white
mottled 4 [Wm4]) that places white next to the heterochromatin of
the X chromosome results in inactivation of this gene during
development in most cells of the eye, although a few small clonal cell
populations still express white (center). The presence of MATH-20 in
eye cells at the correct point in development restores white
expression in the majority of eye cells (at right). Satellite III is a major
component of this heterochromatin the properties of which as a SAR
have been well studied [45], and this 11 megabase satellite is a
target of MATH-20. This targetting presumably interferes with
heterochromatinization, preventing the spread of this repressive
chromatin state and allowing the white gene to acquire and maintain
an active expression state during development.
ditions, chromatids do not form while morphologically
aberrant structures that have a mitotic-like extent of compaction accumulate. Using somatic nuclei, aberrant mitotic
nuclei accumulate which contain highly condensed chromatin pushed against the periphery surrounding an ‘empty’
center (Figure 2b). MATH-20 does not inhibit early mitotic events such as the breakdown of the nuclear
membrane/lamina complex and the disassembly of the
nucleolus. When sperm nuclei are used, biochemical
remodeling and subsequent incorporation of proteins
known to be involved in condensation — such as topoisomerse II [17] and XCAP C/E [18] — are not inhibited by
MATH-20. Interestingly, the structural stability of chromatids in mitotic extracts is also affected (i.e. addition of
MATH-20 results in an ATP-dependent collapse of formed
chromatids into individual spheres [Figure 2b]). It appears
that SAR-bound proteins necessary for the structural stability of chromosomes are displaced to non-SAR sequences by
MATH-20, resulting in a misdirected condensation reaction leading to the formation of chromatid ‘balls’.
Although these observations suggest strongly that SARs are
cis elements of chromosome dynamics, the mechanism
whereby they exert their role remains speculative. SARs
could be the target of binding proteins that either mediate
or facilitate the formation and juxtapositioning of metaphase
chromatin loops. Important recent progress has led to the
identification of a protein complex in Xenopus egg extracts,
called condensin, implicated in chromosome condensation
[19••]. One subunit of this complex is homologous to the
Facilitation of chromatin dynamics by SARs Hart and Laemmli
Drosophila Barren protein, which interacts with topoisomerase II and is required for sister chromatid separation in
mitosis [20]. Topoisomerase II preferentially binds SARs in
vitro, and immunofluorescence and injection studies suggest that this also occurs in vivo [6,21••]. Topoisomerase II
could thus link condensin, via Barren, to SARs whereas
MATH-20 could interfere with this targeting. The aberrant
mitotic structures induced by MATH-20 addition may be a
consequence of such a mistargeting of the condensation proteins to non-SAR sequences.
523
adjacent T3 RNA polymerase promoter (Figure 3a). A
related study [29] found that both the intronic SAR and
associated enhancer of the κ-chain gene are necessary for
the extended, developmentally regulated demethylation
that precedes transcriptional activation of this gene. In this
case, it was shown that SARs from Drosophila or plants
could functionally substitute for the endogenous SAR,
indicating that a general property of SAR DNA rather than
a specific sequence is important [30]. The frequently
observed association of SARs and enhancers [31] might
confer specificity to the initiation of chromatin opening.
SARs and gene expression
Numerous publications have demonstrated that SARs, in
a flanking position, can strongly stimulate the expression
of many but not all heterologous reporter genes
(reviewed in [4]). Remarkably, this activity is evolutionarily conserved. For instance, yeast SARs flanking a
reporter gene increased expression levels 24-fold in
tobacco plant cell lines [22]. The SAR effect is only
observed following stable integration into the genome in
all biological systems tested [22–24]. These cis-acting elements hence appear to require a chromatin
environment as transiently transfected DNA is known to
be poorly organized into nucleosomes. The evolutionary
conservation of the SAR effect on gene expression suggests that conserved structural components of chromatin,
rather than species-specific factors, are involved.
An extended working model [4,25] aimed at explaining the
general stimulatory role of SARs suggests that their clustered A tracts competitively bind two types of proteins.
One type of protein (exemplified in vitro by histone H1 or
topoisomerase II) bind SARs or certain AT-rich satellites
cooperatively and lead to compaction of the chromatin
fiber, chromosome condensation (looping) or heterochromatin formation. Conversely, other proteins such as
HMG-I/Y and the larger derivative MATH-20 bind noncooperatively to SARs and can displace the compacting
proteins by disrupting their cooperative interactions, hence
resulting in chromatin accessibility (or misguided compaction during metaphase). This simple model is based on
in vitro competition experiments demonstrating that
HMG-I/Y and the peptide antibiotic distamycin, which
selectively bind the minor groove of A tracts, specifically
make SAR-containing DNAs accessible by displacing prebound histone H1 from SARs [26]. In vivo support
originates from the observation that distamycin added to
cells increases the accessibility of the internucleosomal
linker DNA of SARs but not of hypersensitive sites [25].
This model is consistent with studies demonstrating that
the developmentally regulated access of the immunoglobulin µ gene utilizes a collaboration between an enhancer
and an associated SAR [27,28••]. In an elegant set of experiments, it was shown that both the SAR and the enhancer
were required to spread the accessibility to a T7 RNA
polymerase promoter located 1 kb away, whereas the
enhancer alone could only confer local accessibility to an
Another example of SAR-mediated gene activation is the
suppression of position-effect variegation (PEV) of the
white mottled allele by the artificial, SAR-specific MATH-20
protein in transgenic Drosophila (Figure 3b; [32••]). This
PEV is caused by a chromosomal inversion that places the
white gene near the centric heterochromatin of the X chromosome. The resulting stochastic inactivation of this gene
is supposedly caused by cooperative spreading of heterochromatin into the juxtaposed euchromatic region.
SAR-like Satellite III sequences are a major component of
this centric heterochromatin [33] and they are a target of
MATH-20. The simplest interpretation follows the model
discussed — that is to say, MATH-20 bound to the
11 megabases of Satellite III sequences is proposed to
interfere with the formation and cooperative spreading of
heterochromatin in this region, thus reducing the number
of cells in which the rearranged white gene has an inactive
chromatin structure.
An alternate model proposes that SAR function is mediated by sequences that readily unwind under torsional stress
[34]. To test this proposal, an oligonucleotide containing an
SAR-derived base-unpairing region was mutated to stabilize base pairing. In contrast to the wild-type
oligonucleotide, the concatenated mutant had reduced
association with nuclear matrices and did not stimulate
gene expression of a chromosomally integrated reporter
gene. These mutations, however, also disrupted A tracts so
this, rather than or in addition to the loss of base-unpairing
potential, could account for the observed effects. Several
proteins have been described that bind to base-unpairing
regions of SARs, apparently as double-stranded DNA
[35•,36–40]. At least some of these are implicated in gene
regulation, are restricted to certain cell types and at least
partially partition into nuclear matrices. Perhaps these proteins target certain SARs to confer specificity to gene
activation similar to the cases described above.
Conclusions
In summary, SARs are cis elements of chromosome dynamics. They play a global role during mitotic chromosome
condensation and are also necessary for the maintenance of
chromosome structure. The reader should note that it
remains to be demonstrated that they are direct binding
sites for protein complexes involved in condensation, such
as condensin.
524
Differentiation and gene regulation
SARs are also implicated in gene expression, by facilitating
both the transition and the spreading of an open chromatin
structure. Another chromatin-specific element might interrupt the spread of chromatin accessibility or inaccessibility
initiated by SARs, thus determining domain boundaries.
Such boundary elements have been shown to restrict
enhancer interactions to intra-domain gene activation
[41–43]. Although the model of SAR function discussed is
well supported by different lines of experiments, however,
other explanations cannot be excluded. For example,
SARs could be preferentially targeted by the nucleosome
remodeling factor CHRAC via topoisomerase II, which is a
subunit of this complex [44].
It is probable that different complexes will be found to target
SARs to achieve dynamic transitions in chromatin structure.
Much work remains to be done to elucidate the relationship
between SAR function and nuclear organization.
Acknowledgements
Work performed in this lab was supported by the Swiss National Fund, the
Canton of Geneva, and the Louis Jeantet Foundation.
References and recommended reading
Papers of particular interest, published within the annual period of review,
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• of special interest
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enhancer plus its associated SAR function together to allow developmentally
regulated gene activation. Here they extend this work by utilizing
bacteriophage T3 and T7 RNA polymerases to probe the accessibility of
chromatin in isolated nuclei. They show that an intronic enhancer can only
open chromatin locally to allow access to an adjacent T3 promoter. In
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