Biology Reference
In-Depth Information
interact with the genome. Thus, binding maps of most NP
proteins cannot be easily interpreted in terms of spatial
positioning of the genome. Nevertheless, FISH experiments
and DamID studies with a NP protein that is exclusively
located at the periphery indicate that some of the genes that
interact with NP proteins are preferentially located at the
nuclear envelope, suggesting that they indeed associate
with NPs. In S. cerevisiae, short sequence elements have
been identified that can target genes to NPs
[40]
.In
Drosophila the NP-associated regions tend to be relatively
short (compared to LADs), but a NP-targeting sequence
motif has not been identified so far
[41]
.
Both in yeast and in fruit flies the interaction of genes
with NP proteins appears to enhance transcription. Inter-
estingly, in yeast activation of the INO1 gene triggers
relocation to the nuclear periphery (presumably to an NP);
this position is retained for several generations after the
gene is shut off. During this period the gene can be turned
on more rapidly, suggesting that NP association facilitates
the swift activation of a gene in response to an external
stimulus
[42]
.
rRNA genes which are transcribed by RNA polymerase I
inside nucleoli, NADs are enriched for specific gene sets,
including olfactory genes and zinc-finger protein encoding
genes. Furthermore, tRNA and 5S RNA genes are prefer-
entially associated with nucleoli, which is of interest
because these genes are transcribed by RNA polymerase
III. Thus, the nucleolus acts as a docking platform for very
specific sets of genes
[43]
.
Interestingly, the nucleolus also appears to keep certain
sequences spatially separated. This was observed for
budding yeast chromosome XII, which harbors the rDNA
gene cluster. Mapping by a 3C-derived method revealed that
DNA sequences at either end of this cluster rarely contact
one another, suggesting that the nucleolus acts as a physical
barrier between the two chromosome segments
[44]
.
THE INTERNAL ORGANIZATION OF
CHROMOSOMES: LONG-RANGE
INTERACTIONS ALONG AND BETWEEN
CHROMOSOMES
The convoluted three-dimensional path of chromatin fibers
is reflected, and in part determined, by multiple specific and
non-specific long-range contacts between and along the
chromosomes. These contact points can be experimentally
detected by direct imaging, but more comprehensively and
at higher resolution, i.e., at the scale of several kb for whole
chromosomes or genomes, using a growing suite of molec-
ular methodologies based on chromosome conformation
capture (3C) technology
[7,45,46]
(
Box 7.2
). Applying such
methods has led to the identification of widespread looping
The Nucleolus as a Spatial Organizer
The nucleolus is a large solid structure inside the nucleus
and hence another prime candidate to act as a scaffold for
chromosome folding. Indeed, a diversity of DNA regions
appear to be associated with the nucleolus. These regions
were identified in human cells by microarray probing and
high-throughput sequencing of DNA that co-purifies with
nucleoli. Like LADs,
nucleolus-associated
domains
(NADs) tend to be large (0.1
10Mb). Besides the 28S
e
BOX 7.2 Chromosome Conformation Capture-Based Technologies
A large variety of chromosome conformation capture
(3C)-based technologies have been developed over the last
decade
[7,45,46]
. All these methods rely on the same strategy
of cross-linking interacting chromosomal loci, fragmenting
chromatin followed by intra-molecular ligation to convert
cross-linked pairs of loci into ligation products to obtain
a genome-wide library of hybrid DNA molecules. Each hybrid
molecule represents a pairwise interaction between the corre-
sponding loci in a single cell. The different 3C-based methods
mainly differ in the way the ligation products are being detec-
ted. The different 3C-based methods are outlined in Figure 7.3.
3C: In a classic 3C experiment the genome-wide ligation
product library is interrogated by quantitative PCR to detect and
quantify the presence and abundance of one ligation product
(chromatin interaction) at the time
[45]
. This low-throughput
approach is used for small-scale and locus-specific studies.
4C: the 4C method employs inverse PCR to amplify all DNA
fragments ligated to a single fragment of interest
[47,48]
.The
amplified DNA is then analyzed on a microarray or is directly
sequenced. This method is used to obtain a genome-wide
interaction profile of a single locus of interest.
5C: the 5C method employs highly multiplexed ligation-
mediated amplification with pools of locus-specific primers to
amplify large sets of targeted chromatin interactions
[49]
.For
instance, all interactions among a set of promoters and a set of
enhancers can be detected in parallel. Amplified interactions
are directly sequenced.
Hi-C: Hi-C is a completely unbiased and genome-wide
adaptation of 3C and differs from the other 3C-based methods
by including a step prior to intra-molecular ligation during
which the ends of the digested chromatin are filled in with
biotinylated nucleotides
[6,22]
. As a result, ligation junctions
will be marked by biotin, facilitating unbiased purification of all
ligation junctions. Purified DNA is then directly sequenced to
obtain a genome-wide chromatin interaction map.
ChIA-PET: The ChIA-PET method employs an immunopre-
cipitation step to enrich for chromatin interactions that involve
loci bound by a protein of interest
[50]
.
