Biology Reference
In-Depth Information
and are then maintained because mixing of long polymers is
an extremely slow process and only occurs at timescales that
are significantly longer than the length of the cell cycle, or
the lifetime of a differentiated cell
[26]
.
A chromosome can fill up a volume that is as small as
the volume of the chromatin fiber itself, which in the case of
our example is only ~40
the nucleus. However, there is considerable evidence that
this assumption is not correct. Instead, specific regions of
the genome appear to be attached to extrachromosomal
scaffolds. This imposes substantial constraints onto the
overall mobility, folding and positioning of chromosomes.
The most prominent scaffold is the nuclear envelope. The
interior surface of the nuclear envelope offers a very large
and heterogeneous area for the anchoring of chromosomal
regions. Indeed, various proteins on the inside of the
nuclear envelope have been implicated in the tethering of
specific chromosomal regions. In addition, the nucleolus
may serve as a docking site.
m
3
, much smaller than the size of
the nucleus, but quite comparable to the size of chromo-
some territories. Within this small volume the chromatin
can fold into a fractal globule state, as has been suggested
for human chromosomes, but also as a confined random
walk or swollen globule, or any mix of these. In any of the
conformations the internal organization of the chromosome
territory would be highly variable between cells, as the
chromatin fiber can follow any random path within the
constrained volume. However, recent studies, described
below in more detail, show that the internal organization is
not random but is further determined by the association of
specific sub-chromosomal domains with nuclear structures
such as the nuclear envelope, and by networks of long-
range interactions between loci leading to formation of
chromatin loops.
The nature of the constraint that can lead to highly
compacted chromatin fibers can be extrinsic or intrinsic: an
example of an extrinsic confinement would be the nuclear
envelope, which can put chromosomes under pressure to
occupy a limited volume. An example of an intrinsic
constraint is the presence of long-range looping interac-
tions between loci along the chromosome. In the following
sections we will discuss the various constraints that act on
the basic conformation of chromosomes dictated by their
polymer characteristics and which modulate their confor-
mation, their internal organization and their subnuclear
positioning.
m
Genome-Wide Techniques to Map Scaffold
Interactions
Two complementary techniques are currently available for
the detection of molecular contacts between the genome
and specific scaffolds. These are chromatin immunopre-
cipitation (ChIP) and DNA adenine methyltransferase
identification (DamID) (
Box 7.1
,
Figure 7.2
). Although
both methods can also be used to map genomic binding
sites of chromatin proteins and DNA-binding factors, we
will only discuss their application for studies of chromo-
some anchoring.
ChIP starts by treatment of cells with formaldehyde,
which rapidly enters the nucleus and cross-links DNA to its
interacting proteins. Contacts between the genome and
scaffold proteins are therefore fixed. After fragmentation of
the nucleus by sonication, a scaffold protein of interest is
purified by immunoprecipitation. DNA sequences attached to
the protein are thus co-purified and can be identified and
quantified using genomic tiling arrays, or by high-throughput
sequencing.
DamID is based on a very different principle. It begins
with the in vivo expression of a chimeric protein consisting
of the scaffold protein of interest fused to DNA adenine
methyltransferase (Dam). This fusion protein is incorpo-
rated into the scaffold. Thus, DNA sequences that contact
the scaffold will also be in molecular proximity to the
ANCHORING OF THE GENOME TO FIXED
SCAFFOLDS
The polymer models described above assume that chro-
mosomes are freely mobile within the spherical confines of
BOX 7.1 DamID and ChIP Methods to Map Genome
Scaffold Interactions
Two complementary methods exist to identify genomic
sequences that are in contact with a nuclear scaffold such as the
nuclear lamina. DamID
[27]
is based on the integration of DNA
adenine methyltransferase (Dam) into a scaffold, by in vivo
expression of a chimearic protein consisting of Dam and
a scaffold protein such as one of the lamins. As a consequence,
any genomic DNA in contact with the scaffold will become
adenine-methylated. This methylation tag, which is not
endogenously present in DNA, can subsequently be mapped by
a series of steps as outlined in
Figure 7.2A
. Adapted from
[7]
.
e
ChIP
[28]
instead employs treatment of cells with a cross-
linking agent such as formaldehyde to covalently attach
scaffold proteins to contacting DNA elements. Next, chro-
matin (with attached scaffold fragments) is isolated, sheared,
and subjected to immuno-purification with an antibody
against a scaffold protein (such as a lamin). The thus obtained
DNA fragments can be analyzed and mapped using genomic
tiling microarrays
or
by
high-throughput
sequencing
(
Figure 7.2B
).
