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Proc
Indian Natn
Sci Acad
73 No.4 pp.
239-253 (2007)
Chromatin
Domain
Boundaries:
Functional
Domains in Genome
239
Research Paper
Chromatin Domain Boundaries: Defining the Functional Domains in Genome
HINA IQBAL and RAKESH K MISHRA*
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
(Received on 31 May 2007; Accepted on 15 October 2007)
Eukaryotic genome is packaged in the nucleus with the help of several proteins. While this packaging is needed to accommodate the large genome within the nuclear volume it also has functional consequences. It is well known that enhancers
can act over a long distance to regulate expression of genes, however, they do not act on inappropriate promoters in the
genome. The elements that prevent such unwanted interactions are called the boundary elements. Boundaries are important for the proper packaging and define functionally independent regulatory domains in the genome. Such elements have
now been identified from yeast to human. Although sequence comparison of such elements does not reveal any similarity,
boundaries from one species can work in other species indicating a functional conservation. This also suggests a common
underlying mechanism of the function of boundary elements. Recent genome sequence information and high throughput
techniques have opened new avenues to understand the nature of boundary elements and the mechanism of their function.
Key words: Chromatin Domain Boundary; Insulator; Barrier; Nuclear Matrix; Genome Organization
Introduction
Eukaryotic genome is chromatinised to be packaged
inside the nucleus with the help of large number of
proteins. The best studied and characterized of all these
proteins are the histones. DNA is wrapped around the
octamer of histones comprised of H2A, H2B, H3 and
H4. The DNA between the nucleosomes, called linker
DNA, is bound by histone H1. This is the first level of
compaction of genome by which 2nm DNA fiber
becomes 10nm “bead on string” leading to a compaction
of six fold. In the next level of compaction nucleosomes
wrap upon themselves to form a solenoid like structure
of 30nm diameter. This 30 nm chromatin fiber gives a
compaction of 40 fold [1-3]. Structural details of
packaging of genome beyond this level of compaction
are poorly understood. Final packaging leading to
10000-fold compaction is required to fit the genome
within the nucleus. This degree of packaging of DNA
in the nucleus is an intricate task and that too in such a
manner that all the nuclear activities can take place
efficiently without any error. Furthermore, each time a
cell divides, its DNA in the nucleus opens up for
replication and after the cell division it is folded back.
While this organization is needed just to accommodate
genome in the nucleus, it is becoming clear that this
packaging has functional consequences as well.
Genomic Packaging and Chromatin Domain
Boundaries
The higher order chromatin organization that ultimately
leads to the chromosome structure is not well
understood. However, various cytological and
biochemical studies have shown that the interphase
chromatin is organized into topologically distinct
domains of varying sizes [4-10]. It was evident from
the electron microscopic studies that chromatin domains
are formed by the looping of 30nm fiber along the
chromosomal scaffold of mitotic chromosome [5, 7]. In
the interphase nucleus, the proteinacious scaffold is in
the form of nuclear matrix and the chromatin domains
are attached to the nuclear matrix through Matrix
Associated Regions (MAR). MAR’s are good candidates
to act as chromatin domain boundaries and at least in
few cases, as discussed below, they have been
shown to possess boundary activity. Chromatin domains
organized in such a manner may also have
functional consequences. In such a case the topological
independence of the domain may coincide with
functionally independent domains of chromatin.
Chromatin domain boundaries are the key structural as
well as regulatory elements that separate these domains,
Fig. 1.
Fig.1: Chromatin domain boundaries: Boundary elements separate structurally and
functionally region of the genome, for example, condensed chromatin from open chromatin
* Address for Correspondence: E-mail: [email protected]
Telephone +(91) 40 27192658; Fax +(91) 40 2716 0951
240
Hina Iqbal and Rakesh Mishra
Chromatin Domain Boundary Assays
2. Enhancer Blocker Assay
Chromatin domain boundaries can be assayed
functionally using transgene-based assay in vivo. Certain
biochemical criterion can also be used to identify
boundaries that can be subsequently tested in a
functional assay. It is possible that all the boundaries
may not have similar properties and hence may respond
differently to different assays. While each of these assays
is useful in identifying putative boundary elements or
studying such elements in a defined region of genome,
characterization of any boundary remains tentative
unless it has been tested to meet at least one of the several
criterion that forms the basis of different assays.
Based on the reasoning used for the insulation from
position effect assay described above, an enhancerblocking assay for DNA segments that can function as
boundaries in vivo have been developed. In this assay
instead of blocking of enhancer and silencer in the
genomic context, test DNA is assayed for its ability to
block a given enhancer from acting on the promoter of
the reporter gene. Accordingly in this assay a test
boundary element is inserted between the enhancer and
promoter of the reporter gene in the transgenic context,
Fig. 3a & b. If test DNA has boundary activity the
enhancer will not be able to act on the promoter and the
reporter gene will be expressed only to a basal level or
not at all depending on the nature of the promoter or
strength of the boundary [12]. Here generally two
reporter genes are used, one for scoring the transgene
and other for testing the enhancer blocking activity of
the test fragment. This is the most commonly used assay
for the boundary elements. Enhancer blocking assay can
also be performed in cell culture system by using
appropriate enhancer promoter combination and may
potentially be used to screen large number of test
fragments for boundary function. In a more recent
version of the enhancer blocker assay the test fragment
is flanked by Lox P sites, which can be used to flip it
out providing the action of Cre recombinase enzyme.
In the case of Drosophila melanogaster Cre expressing
flies lines are available. Transgenic lines carrying the
test DNA flanked by Lox P sites can be crossed with
Cre expressing flies and in the progeny the expression
of the reporter gene can be compared both in the
transgenic and flipped out lines (Fig. 4). Thus in this
assay the expression of the reporter gene can be assayed
both in the presence and absence of the test fragment at
the same genomic location ruling out position effect.
1. Insulation from Position Effect Assay
The expression of any transgene in vivo depends on its
site of insertion in the genome. Often the same transgene
expresses to different levels based on its site of
integration in the genome. This “position effect” is due
to the action of neighbouring regulatory regions on the
transgene. In “insulation from position effect” assay, a
reporter along with its regulatory elements is flanked
by boundary elements on both the sides. Presence of
boundary elements does not allow the regulatory
elements present near the site of integration to act on
the reporter and thus the reporter is protected from the
position effect, Fig. 2. In such a situation expression of
the transgene will be position independent and copy
number dependent. Insulation from position effect was
the first functional assay to test chromatin domain
boundaries in vivo [11]. While it is still very useful, a
drawback of this assay is that a large numbers of lines
are needed to support the conclusions. Flanking with
boundary elements while protects the transgene from
local or position effect, it does not isolate the flanked
DNA from chromosome level mechanisms, for example
the dosage compensation effect in Drosophila [11].
Transgenic lines on the X chromosome typically showed
high level of expression as compared to the lines on
autosomes in males suggesting that boundaries are
unable to protect the reporter gene from the action of
dosage compensation system.
3. Barrier Assay
Heterochromatin is transcriptionally silenced form of
chromatin that can spread into neighbouring
euchromatin. However if a boundary is placed between
Fig. 2: Insulator assay: When a reporter gene is flanked by boundary elements on both the sides it is shielded from the effect of regulatory
elements from outside the domain marked by the boundaries. Both enhancer and silencer cannot act on the promoter of the reporter gene,
as the boundary element does not allow them to act across it.
Chromatin Domain Boundaries: Functional Domains in Genome
241
Fig. 3: Enhancer blocker assay: Boundary element does not allow the enhancer to act on a promoter when placed between the two (a)
and not if it is placed outside the enhancer (b).
Fig. 4: An advanced version of enhancer blocker assay in Drosophila: A boundary element present between the enhancer and promoter
of white gene does not allow the enhancer to act on the promoter resulting in minimal expression of the white gene giving yellow eye
color. When the boundary element is flipped out by bringing in Cre recombinase, the enhancer can act on the promoter resulting in a red
eye color. This assay rules out any position effect.
242
euchromatin and heterochromatin such spreading may
be stalled. This barrier assay is based on this reasoning
that if a heterochromatic region is separated from a
euchromatic region by a boundary element spreading
of silent chromatin is blocked (Fig. 5a & b). In this assay
a reporter gene is placed next to a silenced region
separated from it by a test fragment. If the test fragment
act as a boundary the reporter gene will be expressed
otherwise it will be silenced [13, 14]. This assay has
been used from yeast to vertebrates to test elements that
act as boundaries. In the cell based barrier assay the
reporter gene, usually the test fragment flanks an
antibiotic resistant gene. Cells are selected for antibiotic
resistance following transfection. After selection
antibiotic pressure is withdrawn and the cells are allowed
to grow for several generations. During this time nearby
silenced regions of the genome can spread towards the
reporter gene and repress it. After several generations
antibiotic pressure is again applied. A test DNA with
boundary activity would protect the reporter gene from
repressive heterochromatin allowing the cells to survive
in the antibiotic containing medium [15). This assay is
similar to the insulation from position effect assay in
that it also tests boundaries based on their ability to
protect reporter genes from the effect of nearby
regulatory elements.
Indirect /Biochemical Assays for Boundary Elements
Hina Iqbal and Rakesh Mishra
resulting structures termed halos are digested with
restriction endonucleases and incubated in the presence
of end-labeled DNA probes from the region of interest.
The probes compete with endogenous DNA sequences
for matrix binding. Bound and unbound probe fragments
are separated by centrifugation and analyzed by gel
electrophoresis and autoradiography [16, 17]. In another
version of in vitro MAR assay nuclei are similarly treated
with high salt to extract proteins and digested
exhaustively with DNaseI. DNA and histone depleted
nuclear matrices are incubated with end labeled probe
fragments in the presence of increasing concentrations
of unlabeled competitor DNA. Matrix associated and
unbound probe fragments are separated by centrifugation
and analyzed similarly by gel electrophoresis and
autoradiography [18]. The in vivo MAR assay measures
the partitioning of endogenous DNA sequences between
matrix associated and non-matrix fractions. Similar to
the in vitro assays nuclei are treated with LIS to extract
the proteins and the resulting “halo” is digested with
combination of restriction enzymes. The matrix
preparation is pelleted by centrifugation, and the
partitioning of specific DNA sequences into matrix
associated and non-associated fractions is assessed by
southern hybridization [19]. MAR’s identified from
these methods can than be tested for boundary function
by using transgenic or cell based assays. Often but not
always these MAR’s can act as boundaries [20].
1. MAR Essay
2. DNaseI Hypersensitivity Assay
Chromatin is organized in the nucleus in the form of
loops that form structural domains. Chromatin loops are
attached to the proteinacious scaffold or nuclear matrix
through DNA sequences called MAR. Based on this
property of MAR’s biochemical assays have been
developed to isolate and test MAR sequences. MAR’s
can be mapped both in vitro and in vivo. In case of in
vitro MAR assay nuclei are isolated and treated with
either 2M NaCl or lithium diiodosalicylate (LIS) to
extract histones and other non-histone proteins. The
Chromatin consists of DNA wrapped around
nucleosome. The region of chromatin that is free of
nucleosomes is more accessible and thus hypersensitive
to DNaseI [21, 22]. These sites are generally present in
the regions where other proteins bind and regulate
nuclear processes. Regulatory elements such as
promoters, silencers, and enhancers often map to DNaseI
hypersensitive regions. Most of the known boundary
elements are also associated with DNaseI hypersensitive
sites. Looking for hypersensitive sites, therefore, is a
Fig. 5: Barrier assay: Fragment to be tested for barrier activity is placed between heterochromatin region (a) or silencer (b) and the
reporter gene. If test fragment acts as a boundary heterochromatin will not be able to spread beyond it leaving the reporter gene in
expressed state. Boundary elements can be placed on one side (a) or both sides (b) in this assay.
Chromatin Domain Boundaries: Functional Domains in Genome
way to narrow down a region for new boundaries. In
this assay nuclei are digested with DNaseI for a very
short duration so that DNaseI will cut only in those
regions that are most easily accessible – the
hypersensitive regions. Thereafter DNA is extracted and
digested with restriction endonucleases. As a control
genomic DNA is also digested with the same restriction
endonucleases. Both the control and DNaseI digested
samples are run on agarose gel and probed by the region
of interest. If the region has hypersensitive sites, extra
bands will appear in the DNaseI digested lanes. Using
several restriction enzymes hypersensitive sites can be
precisely mapped in the genome. Once the
hypersensitive regions are identified they can be tested
for boundary function by in vivo assays. This assay,
although not exclusively used for boundary analysis, is
extremely useful in narrowing down of a boundary
element in a suspected region of genome.
3. Chromatin Immuno Precipitation Assay
Few proteins that bind to the boundaries and are
important for their function have been identified [2325]. Chromatin Immuno Precipitation (ChIP) is a
technique used to identify the in vivo binding sites of a
particular protein. The ChIP technique involves using
in vivo cross-linking to stabilize chromatin-associated
proteins to DNA and then to isolate these complexes by
immuno-precipitating them with specific antibodies.
Crosslinks made with formaldehyde can be reversed and
the DNA can be analyzed by PCR or Southern blotting
to determine whether specific DNA sequences are
associated with the protein of interest. ChIP can be
combined with micro array to identify such sites on a
genome level a technique termed ChIP-on-chip [26]. In
this method ChIP is performed as usual and the DNA is
analyzed using a chip of whole genome tiling array. This
technique can be used to identify new regions in a
genome at which a known boundary associated protein
binds. Most of these proteins have multiple functions
and boundary function usually requires binding of more
than one protein. Therefore mere binding of a protein at
a particular region will not classify it as a boundary but
would make it a probable candidate. Based on other
criterion such as neighbouring sequences and binding
sites for other proteins, sequences can be selected and
tested for their boundary function by in vivo assays.
4. Bioinformatic Approach
Sequence comparison of large number of known
boundary sequences gives no sequence similarity. Thus
simple sequence comparison approaches cannot be taken
to find new boundary elements. However, several DNA
sequence motifs to which boundary proteins bind are
known. More than one protein is generally required for
boundary function or several molecules of a protein bind
to a cluster of motifs to function as a boundary. The
clustering of these motifs in the genome, therefore, can
243
be used as an approach to predict boundaries and then
they can be tested using biochemical and in vivo assays.
Such analysis can also be used in a context dependent
manner. For example, boundaries can be predicted based
on the expression pattern of two genes flanking a region.
If the genes flanking a region are differentially
expressed, there is good chance of a boundary residing
in that region. Such predicted boundaries can be tested
using biochemical and in vivo assays. Boundary
elements are known to separate heterochromatin regions
from euchromatin. Such transition regions can be
identified using bioinformatic tools and then tested for
their function. MAR predicting software can be used to
predict MAR’s in a genome and then tested for their
function. In future, a well-annotated genome may
contain boundaries predicted by combining the above
approaches.
Chromatin Domain Boundaries from Different
Organisms (Table 1)
Chromatin Domain Boundaries in Phaseolus vulgaris
The β phaseolin gene in the bean Phaseolus vulgaris is
flanked both on the 3’ and 5’ region by MAR’s [20].
MAR’s have been shown to protect the genes from the
effect of nearby neighboring elements. Earlier studies
have indeed shown that the β phaseolin gene along
with its flanking sequences provides constantly high
and position independent expression in transgenic
tobacco [27]. This result shows that β phaseolin gene is
situated in an independent chromatin loop separated
from the neighbouring environment by MAR’s. Also,
as discussed below, this kind of genome organization
of functionally independent structural domains of
chromatin may be a common feature of genomes of
higher eukaryotes.
Chromatin Domain Boundaries in Saccharomyces
cerevisiae
The yeast Saccharomyces cerevisiae consists of
mating type a or α gene at the mat locus. Identical
copies of these genes are also present at the HML and
the HMR loci but these are transcriptionally silent
(Fig. 6). Insertion of other genes at the HML or HMR
locus renders them silent showing that it is the
property of the locus to remain repressed. The repressed
nature of the HML or HMR is due to the presence of E
and I silencers flanking these regions. Both E and I
silencers have binding sites for a number of proteins
like ACS, Abf1, Rap1. This silencing has been shown
to be dependent on the Sir proteins. The question that
what is it that is not allowing the silencing to spread
beyond HML and HMR led to the hypothesis that
boundary elements present on either side of such
repressed region may prevent spread of silencing in
nearby regions. Such boundaries have indeed been
identified in this region.
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Hina Iqbal and Rakesh Mishra
Table 1. Boundaries in different organisms
Organism
Boundary
Phaseolus vulgaris
3’ and 5’ MAR
Interacting Protein
Motifs at which the protein binds
Saccharomyces
cerevisiae
HMR tRNATHR
TFIIIB, C
Gcn5p
Sas2p
Smc1p, 3p
Tef2 UAS rpg
Rap1p
ACACCCAYACAYYY
Barrier
Tbf1p
Reb1p
TAGGGTT
CGGGTAA
Barrier
Barrier
CHA1 promoter
STAR
Schizosaccharomyces
pombe
Barrier
IRL, IRR
ChIP studies on wild type vs.
deletion strains
Barrier
tRNAALA
Drosophila
melanogaster
scs’
Assays
Insulator
BEAF
D1
CGATA
TTATA
Insulation from position effect
scs
Zw5
GCTGCG
Insulation from position effect
Gypsy
Su(Hw)
Mod(mdg4)
CP190
Topors
PyPuTTGCATACCPy
Enhancer blocker
Fab7
GAGA factor
GAGAG
Phenotype
A6 to A7 homeotic transformation
Fab8
dCTCF
Phenotype
A7 to A8 homeotic transformation
Mcp
Phenotype
A4-A5 homeotic transformation
Sea Urchin
sns
Enhancer blocker
Xenopus laevis
RO
Enhancer blocker
Gallus gallus
5’HS4
CTCF
USF1
CCCTC
CACGGG
Enhancer blocker
Barrier
3’HS4
CTCF
CCCTC
Enhancer blocker
Mus musculus
ICR
CTCF
CCCTC
Enhancer blocker
Homa sapiens
BEAD1
Enhancer blocker
3’ region of CD2 gene
Insulation from position effect
Thr
Tef2 UAS rpg Boundary
tRNA
Boundary
The HML and HMR are silent loci in the yeast
Saccharomyces cerevisiae. Insertion of genes in these
loci renders them silent but when heterologous KanMX
module was inserted at these loci it was expressed. The
R
KanMX module consists of the Kan ORF of E. coli
transposon Tn903 fused to the transcriptional control
sequences of the Tef gene of fungus A. gossypii. It was
found out that it is the property of the Tef2 promoter to
be able to block the silencing from spreading beyond
the silencers (28). When the Aspergillus promoter was
replaced with the Saccharomyces promoter similar effect
was seen further confirming the finding that it is indeed
the property of the promoter to block the spread of
silencing. Subsequent studies showed that the UAS rpg
of Tef2 was sufficient to act as a barrier. The Tef genes
belong to a large family of genes, the ribosomal protein
genes or RPG. Most RPG UAS sites have one or more
binding sites for Rap1. Tef2 UAS has three Rap1 binding
sites, which are critical for barrier function. Such
sequences, however, are not present in the native context
suggesting that the yeast has some other mechanism to
block the spread of silencing [14].
HMR locus is a silenced locus in yeast and the silencing
is due to the presence of two silencers E and I flanking
the HMR (Fig. 6). Insertion of reporter genes at various
positions with respect to the silencers showed that the
silencing effect is restricted within a domain suggesting
that boundary elements flank this domain [13].
Restriction endonuclease accessibility assays also
showed that the HMR domain is flanked on both the
sides by boundary elements. Deletion of the right
boundary led to the spread of Sir3 dependent silencing
[13]. Further it was shown that this element was able to
block the spread of silent chromatin when placed
between the silencer and the promoter of a gene. This
element was also able to block the spread of telomeric
silencing or position effect. Deletion studies of the 1kb
right boundary led to the identification of PolIII
Thr
transcribed tRNA gene and its flanking regions as
responsible for the boundary function [14]. Binding of
TFIIIC to boxB and boxA sequences is the first step in
PolIII transcription. Mutations in the boxB and boxA
sequences compromised the barrier activity suggesting
that this assembly is critical for it. Silencing at HMR is
Chromatin Domain Boundaries: Functional Domains in Genome
245
Fig. 6: Mat locus of Saccharomyces cerevisiae: The silent α- and a- type mating gene are located at the HML and HMR loci, respectively.
Active copies of these genes are located at the MAT locus. The silencing at the HML and HMR loci is due to the presence of E and I
silencers. The right boundary of the HML and HMR loci is mapped to the CHA1 promoter and tRNATHR gene respectively. The HML locus
is ~2.5 kb whereas the HMR locus is ~1.6 kb. The active MAT locus is ~2.5 kb.
dependent on Sir2, a protein that possess deacetylase
Thr
activity. Barrier activity of tRNA was compromised
in SAS2 and GCN5 mutants [29]. Several other PolIII
transcribed genes showed no barrier activity when
checked for it. Both the silencer and barrier at HMR I
were rich in Sir3 whereas the region after the barrier
was rich in active marks such as histone acetylation and
methylation at H3K4, H3K36 and H3K79. Also the
barrier element was partially depleted of nucleosomes
in accordance with the fact that PolIII initiation complex
excludes nucleosomes and positions them on either side
of tRNA. In a strain devoid of tRNA the nucleosome level
at the barrier was comparable to the sites adjacent to it,
suggesting that binding of the PolIII initiation complex
to the tRNA is necessary for nucleosome exclusion. At
another locus it was shown that nucleosome “hole”
(region deprived of nucleosomes) can function as a
barrier [14], but at this locus reappearance of
nucleosomes led to only a marginal increase in the
recruitment of Sir proteins suggesting that though
nucleosome “hole” might be necessary it is not sufficient
for barrier function. Above studies suggest that both
chromatin modifying enzymes and tRNA play important
role in barrier activity [29].
CHA1 Promoter
CHA1 gene is present 2kb from the HML I silencer (Fig.
6). In normal conditions it is repressed due to the
silencing effect of HML I in Sir4 dependent manner [30],
but when serine is added to the media CHA1 promoter
is able to overcome the effect of silencing. This result
suggested that a boundary element may be separating
the CHA1 gene from the HML I silencer and that this
boundary function is based on serine induction. This
was indeed found that the CHA1 promoter along with
the UAS sequences was able to act as boundary in the
barrier assay when serine was added to the medium and
this boundary function is lost in the medium lacking
serine [31,32].
Sub Telomeric Anti Silencing Regions (STAR’s)
Telomeric regions are heterochromatic in nature. Yeast
telomeres have repeats of (TG1-3)n, followed by up to
four Y’ repeats which are followed by X repeats [33].
The 6.7 kb Y’ element is highly conserved. The X repeats
are heterogeneous both in size (0.3-3.75) kb and
sequence compared to the Y’ repeats. Internal to these
elements are other repeat elements which are then
followed by sequences unique to each chromosome [34,
35]. In order to understand whether these X and Y’
regions have any role in stopping the spread of
heterochromatin from the telomeres, a reporter gene
(URA3) was inserted at different regions in the Y’ repeat
and X repeat. It was found that 0.3 kb of the STR (sub
telomeric region) from the X repeat and 0.14 kb of the
Y’-STR were able to act as barriers to the spread of
heterochromatin. These regions were thus referred to
as STAR’s (Sub Telomeric Anti Silencing regions) [36].
Chromatin Domain Boundaries in Schizosaccharomyces pombe
Inverted Repeat Left (IRL) and Inverted Repeat Right
(IRR)
In fission yeast mating type region contains three linked
loci, viz., mat 1, mat2, and mat3. The mat1 locus is
transcriptionally active whereas the mat2 and mat3 loci
and the region between them also known as the K region
are transcriptionally repressed (Fig. 7). ChIP analysis
showed that mat 2 and mat 3 loci and the region between
them are enriched in swi6, a homolog of Drosophila
9
HP1, and H3 Lys methylation mark. These feature
decreases sharply on the either side of the repressed mat
9
locus. The peak of H3 Lys methylation before it declines
on either side matches exactly with two inverted repeats
IRL and IRR. Also it was shown that the marker genes
were repressed when inserted inside the mat2/3 loci
flanked by the repeats but not when inserted outside of
4
the repeats. H3 Lys methylation is a mark of active
chromatin and the mat2/3 interval flanked by the
inverted repeats was found lacking this active histone
mark whereas the region outside the repeats was
enriched with this mark. Upon deletion of these repeats
9
swi6 and H3 Lys methylation enrichment was found
extended beyond the mat2/3 locus. These repeats thus
function as boundaries of the mat2/3 locus [37]. The IR
elements contain multiple B boxes to which the
transcription factor TFIIIC binds and this binding was
246
Hina Iqbal and Rakesh Mishra
Fig. 7: Mat locus of Schizosaccharomyces pombe: Fission yeast mating type region contains three linked loci mat 1, mat2, and mat3
spread to about 34 kb. The mat1 locus is transcriptionally active whereas the mat2 and mat3 locus and the region between them also
known as the K region are transcriptionally repressed. IRL and IRR are inverted repeats that separate the transcriptionally active region
from the transcriptionally repressed region.
found to be necessary for the boundary activity. PolIII
is not recruited at the IR’s but they are transcribed. IR’s
have binding site for PolII subunit Rpb1 and it is
suggested that PolII might be responsible for the
transcription of IR’s. In the genome of
Schizosaccharomyces pombe at many other sites also
there is enrichment of TFIIIC without any enrichment
of PolIII binding. These sites are termed COC
(Chromosome-Organizing Clamps) sites and may have
boundary function. Immunofluorescent staining showed
that Sfc3 and Sfc6, subunits of TFIIIC, were highly
concentrated at five to ten bodies present at the nuclear
periphery. TFIIIC associated sites including the mat and
COC sites were also found to be present at the nuclear
periphery suggesting that these sites may have a role in
organizing the fission yeast genome [38].
tRNAALA Barrier
The centromeres in Schizosaccharomyces pombe consist
of a central core cnt region followed by the inner repeats
and then the outer repeats on both the sides (Fig. 8).
The nucleosomes at the central core chromatin are
methylated at H3 Lys 4 whereas the outer repeat
nucleosomes are methylated at H3 Lys9 and are bound
by HP1 homolog Swi6. Inner repeats (imr) form the
transition region between these two distinct types of
chromatin suggesting that this region has barrier function
which prevents the outer repeat heterochromatin to
encroach into central core region. The inner repeats
consist of several tRNA genes. A recent report showed
that indeed the tRNAALA functions as a barrier [39]. tRNA
gene promoter contains box A and box B sequences as
well as upstream TATA sequences. Alteration in the
sequence between box A and box B had no effect on
barrier activity whereas mutations in box A sequences
eliminated barrier activity suggesting a role of RNA
PolIII transcription assembly in barrier function.
Chromatin Domain Boundaries in Drosophila
melanogaster
scs and scs’
The first elements possessing boundary function were
identified by biochemical means in Drosophila. These
regions known as scs (specialized chromatin structures)
and scs’ flank the two divergently transcribed hsp70
genes at the 87A7 region [40]. Each boundary element
has a pair of closely spaced nuclease hypersensitive sites
arranged around a central nuclease resistant core. Both
scs and scs’ have shown to block enhancer promoter
interaction in transgenic assays [11]. Although both scs
and scs’ function as chromatin domain boundaries they
do not show any sequence similarity. BEAF (Boundary
Element Associated factor) binds to scs’ and is needed
for its boundary function. BEAF has two isoforms 32A
and 32B that have common carboxy terminal domain,
which is needed for protein-protein interaction but
different N terminal domain essential for interaction with
DNA [24]. BEAF 32A and BEAF 32B bind to the scs’
element as a trimer, 32A-32B 2 [41].In polytene
chromosomes BEAF antibody stains a large number of
interbands suggesting its role in demarcating the
boundaries of independent domains. Zw5 protein binds
to the scs element and is essential for its enhancer
blocking activity [25]. Mutations in zw5 gene affect the
boundary function of scs. BEAF and Zw5 interact with
each other [42] suggesting that this interaction might
facilitate pairing of scs and scs’ elements leading to the
partitioning of chromatin into autonomous functional
units.
Gypsy Retrotransposon
Gypsy is a retrotransposon that gets inserted in the
Drosophila genome at several positions. One such
insertion of gypsy is in the start site of yellow gene.
yellow gene is responsible for bristle, wing and body
pigmentation in Drosophila. Enhancers controlling
yellow gene in the wings and body cuticle are located in
the 5’ region of the gene whereas those controlling
yellow expression in the bristles are located in the intron
of the yellow gene. Gypsy inserted at –700 bp from the
transcription start site in one of the alleles of yellow, y2,
leads to a phenotype consisting of mutant wing blades
and body cuticle but wild type bristles in the adult fly
[43]. Insertion of the gypsy in the 5’ region of yellow
inhibits the interaction of the upstream wing and body
cuticle enhancers with the promoter but does not affect
the function of bristle enhancer located downstream to
the promoter. Thus gypsy retrotransposon acts as a
boundary that separates wing and body enhancers from
the promoter of the yellow gene. Mutations in the
Chromatin Domain Boundaries: Functional Domains in Genome
247
Fig. 8: Centromere1 of Schizosaccharomyces pombe: Central core (cnt1) is flanked by inner repeats imr1L and imr1R. Outer repeats
otr1L and otr1R flank the inner repeats. Inner repeats contain six tRNA genes that are denoted by black bars. tRNAALA genes are denoted
by asterisk. The region shown is ~40 kb.
suppressor of Hairy-wing, su(Hw), gene reverse gypsy
induced mutations, suggesting that this protein plays a
central role in mediating gypsy effects on transcription
[44]. This gene encodes a protein with set of 12 DNA–
binding ‘Zn finger’ motif. It was found that mutations
in modifier of mdg4, mod(mdg4), enhance the effect of
su(Hw) suggesting an interaction between the two
proteins. The mod(mdg4) protein controls the nature of
the repressive effect of su(Hw): in the absence of
mod(mdg4) protein, su(Hw) exerts a bi-directional
silencing effect, whereas in the presence of mod(mdg4),
the silencing effect is transformed into unidirectional
repression [45]. A genetic screen for dominant enhancers
of mod(mdg4) resulted in the identification of CP190
as the third component of the gypsy insulator [46].
CP190 associates physically with both su(Hw) and
Mod(mdg4) and co localizes with both proteins on the
polytene chromosomes. Mutations in the CP190 gene
impair the function of the insulator present in the gypsy
retrotransposon without affecting the presence of su(Hw)
and Mod(mdg4). A yeast two-hybrid screen for proteins
that interact with Mod(mdg4) resulted in identification
of dTopors (Drosophila Topoisomerase I-interacting RS)
protein. dTopors was found to interact with three known
insulator components su(Hw), mod(mdg4) and CP190
and is required for gypsy insulator function. Over
expression of dTopors in the mod(mdg4)2.2 null mutant
rescues insulator activity. dTopors associates with the
nuclear lamina, and mutations in lamin disrupt dTopors
localization as well as nuclear organization and activity
of the gypsy insulator [47]. Recently, it was shown that
gypsy insulator activity is decreased when Argonaute
gene required for RNAi, is mutated, while the insulator
function is improved when levels of Rm62 helicase,
involved in ds-RNA mediated silencing and
heterochromatin formation, are reduced. Rm62 was
found to interact physically with the DNA- binding
insulator protein CP190 in an RNA-dependent manner
[48]. These observations suggest a functional link
between boundary and RNAi system although much
remains to be done to understand the nature of this link.
Boundaries of the Bithorax Complex
The BX-C contains three homeotic genes, Ubx, abd-A
and Abd-B that are responsible for the identities of
parasegments that form the posterior half of the thorax
and abdomen. Precise parasegmental expression patterns
of these homeotic genes are crucial for generating a
normal body plan, and misregulation of these genes
result in dramatic transformation of one body segment
into another [49, 50]. The expression of Abd-B is
controlled by five regulatory regions iab-5 to iab-9 in
parasegment specific manner (Fig. 9). These regulatory
regions are separated from one another by boundary
elements. One of these boundaries, Fab-7
(Frontoabdominal-7) is situated between iab-6 and iab7. Mutation in the Fab-7 region results in a gain of
th
function phenotype - a transformation of 6 abdominal
th
segment to 7 [51-53]. This transformation appears to
be due to the inappropriate activation of the iab-7 cisregulatory region in more anterior region of the embryo,
where Abd-B is normally controlled by iab-6. Fab-7
function is dependent on the GAGA factor which binds
to GAGAG motif [54]. Similarly Mcp and Fab-8
boundaries are responsible for the regulation of Abd-B
expression in different part of developing embryo [55,
56]. Mcp is situated between iab-4 and iab-5 and
th
mutation in Mcp results in the transformation of 4
th
abdominal segment to 5 . Fab-8 is situated between iab7 and iab-8 and mutation in this boundary results in the
th
th
transformation of 7 abdominal segment to 8 . Recently,
this boundary has been shown to be dependent on
Drosophila homolog of CTCF [57] a well known
boundary interacting protein in mammals. These
observations suggest a key role of boundary elements
in the organization and regulation of hox gene clusters.
Chromatin Domain Boundaries of Sea Urchin
sns (Silencing Nucleoprotein Structure)
The sea urchin histone-repeating unit has one copy each
of the five histone genes whose coordinated expression
during development is regulated by gene-specific
elements. Although only one transcriptional enhancer
described as the modulator element of the H2A gene is
identified in this unit, each gene within the repeat is
apparently regulated by gene-specific transcriptional
elements. It was hypothesized that there might be
boundary elements that would restrict H2A modulator
function to its cognate promoter. A DNA fragment
downstream of the H2A gene was bound to posses
enhancer blocking activity in transgenic sea urchin
embryos [58, 59]. This element defined as sns (silencing
248
Hina Iqbal and Rakesh Mishra
Fig. 9: Abd-B region of the bithorax complex of Drosophila melanogaster: The expression of Abd-B is controlled by five regulatory
regions iab-5 to iab-9. These regulatory regions are separated from one another by boundary sequences, mutations in which
result in dominant phenotype which can be explained by ‘inter mixing of otherwise independent regulatory domains’. The region shown
is ~140 kb.
nucleoprotein structure) was later shown to work as
enhancer blocker in human cells as well [60] suggesting
that the trans acting factors required for its boundary
function might be conserved across vertebrates.
Chromatin Domain Boundaries of Xenopus laevis
Repeat Organizer
The rRNA genes of eukaryotes are organized in tandem
arrays in which the genes are separated by intergenic
spacers. The intergenic spacer of rRNA genes in Xenopus
is composed of repetitive sequence elements. The
repeats are of different kinds – 35bp repeats, 60bp
repeats, 81bp repeats and 100bp repeats, taq boxes and
spacer promoters. The function of all the repeats is not
clear. In an attempt to functionally characterize these
repeats, a plasmid construct carrying tandem repeat of
rRNA reporter genes separated by the 35 and 100 bp
repeat region and the rRNA gene enhancer was injected
into Xenopus embryos and the expression of reporter
genes was studied. When the intergenic spacer region
is present in its normal position and orientation
downstream of the rRNA reporter genes, the enhancer
activates the adjacent downstream promoter but not the
upstream rRNA promoter on the same plasmid, thus
functioning like an enhancer blocker. This intergenic
spacer region defined as RO (Repeat Organizer) is
different from the usual enhancer blockers in that to act
as enhancer blocker it needs to be in same position and
orientation with respect to other sequence elements of
the rRNA genes. RO, therefore, is a specialized insulator
element (61).
Chromatin Domain Boundaries of Chicken
Boundaries of the β Globin Locus
The chicken β globin locus contains four globin genes.
Upstream of this locus is the Folate Receptor gene and
downstream is the Odorate Receptor gene. Downstream
of the FR gene and upstream of the β globin locus is a
region of condensed chromatin. Several DNaseI
hypersensitive sites were identified upstream of this
locus (Fig.10). The first vertebrate boundary element to
be identified was the 5’HS4 (DNaseI Hypersensitive
sites) of the chicken β globin locus which stops the
spread of condensed chromatin into the β globin locus
[15, 62]. A 1.2 kb region from this element is able to
prevent enhancer promoter interaction in a transgenic
assay using human cell line. Dissection of this region
showed that a 250 bp core region is sufficient for the
enhancer-blocking activity (Chung et al., 1997). CTCF
binds to this region and is necessary and sufficient for
the enhancer blocking activity [23]. Subsequent studies
showed that the endogenous 5’HS4 sequences are highly
4
enriched in histone acetylation and H3 Lys methylation.
CTCF is not responsible for these modifications, and
another protein, USF1, is responsible for the recruitment
of histone modification machinery. Thus the 5’ HS
element of the chicken ² globin locus possess two
properties one of enhancer blocker and the other of
barrier both attributed to different proteins binding at
separate sites in the element [63, 64]. At the other end
of the locus, 3’HS region was also found to be capable
of CTCF dependent enhancer blocker function [65].
Fig. 10: Schematic representation of the chicken β globin locus: The chicken β globin locus contains four globin genes. Upstream of this
locus is the Folate Receptor (FR) gene and downstream is the Odorate Receptor (OR) gene. Upstream of the β globin locus is a region of
condensed chromatin. Several DNAseI hypersensitive sites (denoted as arrow marks) separate ² globin locus from condensed chromatin.
The 5’ HS4 (hypersensitive site 4) acts as a boundary separating the condensed chromatin from the active region of the globin locus.
Towards the 3’ end of the goblin locus another HS is present. This 3’HS acts as a boundary and does not allow the enhancer (denoted by
blue oval) of globin locus to act on the OR gene. The region shown is ~30 kb.
Chromatin Domain Boundaries: Functional Domains in Genome
Boundaries of the Chicken α Globin Domain
The chicken α globin gene locus consists of several
genes and cis regulatory elements. Many DNaseI
hypersensitive sites both tissue specific and constitutive
are present upstream of the locus. Comparing the
situation with the β globin locus it was thought that they
may act as boundaries of this locus. Two of these sites
when checked for enhancer blocker activity in cell
culture assay indeed turned out to be CTCF dependent
boundary elements [66].
Chromatin Domain Boundaries of Mouse
Imprinting Control Region (ICR)
The most striking example where an insulator element
is directly involved in gene regulation is that of the ICR
element controlling the imprinted expression of H19 and
Igf2 [67-69]. At the Igf2/H19 locus in mouse, rat and
human, the Igf2 gene is expressed only from the
paternally derived allele and H19 only from the maternal
allele. The paternal allele is methylated in a region (the
imprinted control region or ICR) that lies between the
two genes. The ICR contains an insulator element at
which the CTCF protein binds (Fig. 11). In the maternal
allele ICR is non-methylated so CTCF binds to it leading
to its insulator function and thereby not allowing the
downstream enhancers to act on the Igf2 promoter. The
enhancer in this situation drives H19 from the maternal
249
allele. In the case of paternal alleles because the ICR is
methylated CTCF cannot bind at it leading to loss of
insulator function. Now the downstream enhancer is free
to activate Igf2. H19 is silenced because of methylation
at the ICR that spreads up its promoter.
Chromatin Domain Boundaries of Human
Blocking Element Alpha/Delta-1 (BEAD-1)
α, β, γ and δ genes encode T Cell Receptor (TCR)
proteins at the TCR locus. This locus contains a large
number of cis regulatory elements. Due to the close
apposition of differentially regulated gene segments
within the TCR α/δ locus, it was thought that boundary
elements might be present in this locus. TCR α and δ
genes are differentially expressed although they are
located in close proximity at the same locus. A 2 kb
element situated between the TCR δ gene and the
promoter of the TCR α gene when tested for boundary
function in cell-based assay was able to act as enhancer
blocker. This element termed BEAD-1 (Blocking
element alpha/delta-1) in its native context insulates the
promoter of the T α gene from the Eδ enhancer situated
upstream [70]. However, when tested for boundary
function in Drosophila melanogaster it failed to block
enhancer promoter communication suggesting that the
factors required for its boundary function are missing
in fly [70].
Fig. 11: The Igf2/H19 Imprinting Control Region (ICR) of mouse: The Igf2 and H19 genes in mouse are separated from each other by a
region containing ICR which has binding site for CTCF. Enhancers downstream of H19 regulate both the genes. In the maternal allele
CTCF binds to the ICR leading to its insulator function and thereby not allowing the downstream enhancers to act on the Igf2 promoter
and thus only H19 is expressed from the maternal allele. In the case of paternal alleles ICR is methylated so CTCF cannot bind to it
leading to loss of insulator function. This allows the enhancer to activate Igf2. The region from Igf2 to H19 is ~90 kb.
250
Hina Iqbal and Rakesh Mishra
3’ Region of the Human CD2 Gene
Functional Conservation of Boundaries
CD2, a 55 kDa glycoprotein, is a marker for
differentiating T cells. A 28.5 kb genomic fragment
containing five exons of the human CD2 gene as well
as 4.5 kb upstream and 9 kb downstream flanking
sequences when introduced into the genome of
transgenic mice conferred high levels of expression of
the transgene on the surface of T cells and
megakaryocytes. Expression of the transgene was
independent of the site of integration and copy number
dependent suggesting that locus control regions are
present upstream and downstream of the CD2 gene that
determine high levels and tissue specific expression.
Biochemical analysis of the thymus nuclei isolated from
these transgenic mice showed DNaseI hypersensitive
sites at the promoter of the CD2 gene as well as towards
the 3’ end. This suggested that these regions might be
acting as boundaries and shielding the CD2 gene in T
cells from the effect of neighbouring regulatory
elements. To confirm the above speculation a transgenic
construct of CD2 was made without the downstream
hypersensitive site. This construct showed no tissue
specific expression confirming that the 3’ hypersensitive
sites are needed for the T cell specific and copy number
dependent expression of CD2. Finally, a fusion construct
of CD2 3’ region with the Thy-1 gene or the human βglobin gene resulted in T cell specific and copy number
dependent expression of the transgene. These results
confirmed that human CD2 3’ flanking sequences
function as boundary elements and confer high-level, T
cell-specific, position independent expression in
transgenic mice [71].
At present there is lot of interest in the study of chromatin
domain boundaries because while it clear that such
elements are key components of chromatin organization
in all eukaryotes the mechanism of their function is not
clear. A further level of complexity is added by the
observation that when two gypsy elements are placed
next to each other they cancel the boundary function on
one another [74]. There are, however, observations that
that a boundary element can functionally replace another
one. For example, both gypsy and scs’ were able to
replace Fab-7 in its native context showing that there is
underlying general mechanism by which many
chromatin domain boundaries function [75]. When
boundary element from one organism is tested for its
function in another organism they often work. Gypsy
retrotransposon functions as an enhancer blocker in yeast
[31], and mouse boundary element is able to block
enhancer promoter communication in transgenic
Drosophila melanogaster (D. Vasnathi and RK Mishra,
personal communications). Work from our lab has also
shown that repeat regions from human are able to act as
enhancer blocker in Drosophila melanogaster (RP
Kumar and RK Mishra, personal communications). All
these results suggest that there is enough conservation
of both cis elements and the trans acting factors that
boundary element from an organism can function in
evolutionarily distant relatives.
Sequence Requirement and Abundance of Boundary
Elements
No sequence similarity is known among different known
boundary elements. This lack of sequence similarity
among the different boundaries makes the search for
unknown elements difficult by means of simple
sequence comparison approaches. Even the size of
known boundaries varies a lot, although most of them
are few hundred bases. As quite a few proteins, which
interact with the boundaries, are now known, one
approach to study new boundaries would be to search
for the targets of such proteins at genome level implying
techniques such as ChIP-on-chip. Since chromatin
domain boundaries are important component of the
higher order chromatin organization, it is expected that
there should be large number of such elements present
in the genome. If we consider the average domain size
to be 60 kb [7, 72] the human genome should consist of
50 thousand boundaries. A higher density of boundaries
would be expected in transcriptionally busy regions of
the genome. Biochemical studies have mapped boundary
associated proteins such as BEAF and su(Hw) [24, 73]
to a large number of sites in the Drosophila melanogaster
genome further strengthening this view.
Mechanism of Boundary Function
Chromatin domain boundaries are known for sometime
but we have started to understand the mechanism of their
function only very recently. These elements are
identified by their two hallmark properties. One is to
confer position independent expression of a transgene
and other is to restrict enhancer promoter
communication only when placed between them (Fig.
2, 3). Thus boundaries are neutral structural elements
that prevent cross talk between flanking regulatory
elements. In order to understand the mechanism of
function of boundaries it is important to understand how
regulatory elements such as promoters, enhancers,
silencers and repressors work. Boundaries are DNA
elements that function by recruiting different proteins.
Only a handful of boundary proteins are known which
are all DNA binding. These include BEAF, Zw5,
Su(Hw), GAGA factor and CTCF [24, 76], of which
CTCF is the only known vertebrate boundary protein.
By immunostaining and ChIP-on-chip technique it has
been shown that these proteins bind to hundreds of sites
in the genome. For some of these proteins other
interacting partners are also known, which gives a clue
that boundaries function by recruiting some proteins and
in turn these proteins interact with other factors in cell
type specific manner, so that the same DNA element
can function as a boundary or not in a particular cell
Chromatin Domain Boundaries: Functional Domains in Genome
type depending on the presence or absence of the
transacting partners.
Due to their ability to block enhancer promoter
communication one early view about the mechanism of
function of boundaries was that they act as “promoter
decoys”. But this view was soon discarded because if
boundaries acted as “promoter decoys” then why they
should block enhancer promoter communication only
when placed between the two. This hypothesis fails to
explain why an insulator placed of either side to enhancer
does not disrupts its interaction with the promoter. One
current hypothesis about the mechanism of boundary
function is based on the supposition that enhancers act
on promoters via some signal that traverses from
enhancers to promoters, and boundaries act as barriers
to this signal (Fig. 12). This signal could be in the form
of some protein molecule or the RNA PolII itself. The
barrier hypothesis gets support from the experiments
done in yeast. Almost all the boundaries identified in
yeast act as barriers to heterochromatin spread.
Heterochromatin is the condensed form of chromatin
characterized by the presence of repressive marks on
9
histone tails such as methylation of Lys on Histone H3
and lack of acetylation marks. These modifications are
brought about by histone methyltranferases and histone
deacetylases that are recruited in the heterochromatic
regions. When a contiguous array of nucleosomes is
present these enzymes can modify the tails of
euchromatic histones rendering the environment
heterochromatic. However, when the nucleosome array
is interrupted the heterochromatin will not be able to
spread beyond that. This contiguous spread can,
therefore, be interrupted by the repositioning of
nucleosomes in such a way that a nucleosome “hole” is
created. Such nucleosome “hole” has been shown to
function as a boundary as discussed earlier [14]. But
this is a case of a simple eukaryote where the
heterochromatin is only a tiny component. In higher
eukaryotes where the heterochromatin is a major
component of the genome and stretches up to several
Mb, the possibility of nucleosome “hole” to function as
a boundary is unlikely. In higher eukaryotes this
contiguous array of nucleosomes can be broken by the
presence of multiprotein complexes sitting on the
251
chromatin. According to this view boundaries work by
recruiting large multiprotein complexes thereby not
allowing the heterochromatic enzymes to move into
euchromatin.
Yet another hypothesis about boundary function is
that boundaries work by structurally compartmentalizing
the chromatin into looped domains and that these
structural domains also coincide with the functional
domains of chromatin. If an enhancer and promoter lie
in the same loop there are more chances of enhancer
promoter communication whereas if the promoter lies
outside the loop then the chances of enhancer acting on
that promoter are less (Fig. 13). This hypothesis has
gained much acceptance due to some elegant work done
by several labs. In one of the first reports supporting
this view was that scs and scs’ interact in vivo forming a
15 kb looped chromatin domain that includes the two
87A7 hsp70 genes and this interaction is mediated by
the insulator proteins Zw5 and BEAF which bind to scs
and scs’, respectively [42]. Recently, work from our lab
has shown that BEAF is a component of the nuclear
matrix and that BEAF and Zw5 co localize in the nuclear
matrix preparation along with scs’ element [77]. In
another report it was shown that gypsy insulator bodies
are formed by the association of Su(Hw) proteins
forming looped domains [78]. Additionally these looped
domains are tethered to the nuclear periphery via the
interaction of Mod(mdg4) with dTopors [79]. Tethering
to a sub nuclear surface has also been shown in the
case of chicken β-globin insulator. In this case
tethering of the insulator site to the nucleolar periphery
is brought about by the interaction of CTCF with
nucleophosmin resulting in the formation of looped
domain structures [80].
Very recently a SINE B2 repeat has been shown to
work as a boundary and this boundary function is
dependent on bidirectional transcription from PolII and
polIII promoters [81]. Thus it is clear that boundaries
function in more than one way and employ different
mechanisms for their function, although most of the
boundaries seem to make use of looping mechanism,
creating domains of independent structural and
functional identity. Boundaries are not the only means
of higher order chromatin mediated transcriptional
Fig. 12: Enhancer drives the promoter by a signal, which traverses from the enhancer to the promoter. Boundary elements block this
signal thereby not allowing the enhancer to act on the promoter.
252
Hina Iqbal and Rakesh Mishra
Fig. 13: Boundary elements are attached to the nuclear matrix bringing the regulatory sequences into a nuclear compartment. Enhancer
promoter interactions are more feasible in a nuclear compartment as compared to enhancer promoter interactions between neighboring
compartments. Blue oval represents enhancer, red arrows denote promoters and group of small ovals represent structural proteins that
mediate interaction of boundaries with the nuclear matrix.
regulation. One such mechanism shown at the murine
β-globin locus is the formation of Active Chromatin hub,
ACH [82]. At this locus it was shown that different
hypersensitive sites and active genes physically interact
in the nuclear space whereas the inactive genes loop
out. This spatial unit where different regulatory elements
come together for proper transcriptional regulation was
termed “ Active Chromatin Hub”. It is possible that
chromatin is partitioned into structurally and
functionally independent units by boundaries and within
these units ACH like mechanism may be operating to
make appropriate enhancer choice. Thus boundaries
work at a higher level in the organization of chromatin
creating regulatory compartments and features such as
ACH may be formed by the local looping of chromatin
based on preferential enhancer promoter interaction in
such compartments.
Boundaries may separate active compartments from
repressive ones and create a dynamic equilibrium in the
nucleus. Different regions of chromatin can approach
from repressive compartment to active and vice versa
by boundary dynamics. For example, two boundaries
can interact with each other and bring a region from
repressive to active compartment. One of the major
difficulties in detail molecular analysis of the boundary
elements is the lack of direct biochemical assays. Only
transgene based in vivo assays are available which limit
the large-scale studies. With the recent development,
now by ChIP-chip approach the targets of these proteins
can be analyzed at whole genome level. It will be
unrealistic, however, to assume that all such targets will
work as boundaries or all boundaries can be identified
by this approach. Thus new assays need to be developed
to study chromatin domain boundary elements on large
scale and understand the mechanism of their function.
Acknowledgements
Work in RKM laboratory has been supported by Human
Frontier Science Program Organization (HFSPO), IndoFrench Centre (CEFIPRA), Department of
Biotechnology (DBT, Govt. of India) and the Council
for Scientific and Industrial Research (CSIR, Govt. of
India). HI acknowledges a fellowship from CSIR.
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