Biology Reference
In-Depth Information
interactions between loci separated by large genomic
distances, sometimes up to several Mb. Networks of such
interactions may be involved in gene regulation and
constrain the path of the chromatin fiber within chromosome
territories in a cell type-specific manner.
concept of 3C, but differ in the way chimeric molecules are
detected (
Box 7.2
;
Figure 7.3
). For instance, the Hi-C
variant includes a step to mark ligation junctions with
biotin to facilitate their purification, followed by deep
sequencing to generate a long-range chromatin interaction
map for a complete genome
[22]
.
3C-based data provide insights into the probability, or
frequency in the cell population, that any two loci are
physically touching, and thus has become the method of
choice to map the average spatial path of the chromatin
fiber in a cell population.
Molecular Techniques to Map Long-Range
Chromatin Interactions
3C-based technologies are used to detect pairs of loci
located on the same chromosome or on different chromo-
somes that are in close spatial proximity
[45]
. Cells are
cross-linked with formaldehyde to covalently link
segments of chromatin fibers that are physically touching or
interacting through co-binding proteins or subnuclear
structures. This leads to linking of pairs of interacting loci
throughout the genome. Chromatin is then solubilized and
fragmented in small segments using a restriction enzyme to
generate a complex mixture of linked pairs of restriction
fragments. DNA is then intra-molecularly ligated so that
pairs of interacting loci are converted into unique chimeric
DNA molecules. Chimeric molecules, each representing
a physical contact between a pair of chromatin segments,
can then be detected and quantified by PCR, or more
comprehensively by deep sequencing. The frequency with
which a specific chimeric DNA molecule is detected is
typically interpreted to represent the fraction of cells in the
cell population in which a specific pair of loci is in close
spatial proximity.
Over the years a large variety of 3C-based methods have
been developed
[7,46]
. All of these rely on the basic
Determination of Polymer Parameters using
3C Chromatin Interaction Data
As outlined above, the contact probability of a chromatin
fiber is expected to decrease for loci located at increas-
ingly large distances along the chromosome, with the
precise function of this decay related to the polymer state
of the chromosome. 3C-based interaction analyses have
been used to study this relationship in detail. For yeast
chromosomal arms it was found that the contact proba-
bility of loci scales as P(s)~s
-3/2
, consistent with a random
walk conformation
[45]
. Further studies that combined 3C
assays and measurements of 3D distances between loci
allowed estimation of the mass density and persistence
length of yeast chromatin
[18]
.Themassdensitywas
found to be around 40 nm/kb, much longer than would be
predicted for a canonical 30 nm fiber, and the persistence
length was found to be around 95 nm, corresponding to
2.3 kb.
FIGURE 7.3
Chromosome conformation
capture-based technologies. See box 7.2 for
further description of these technologies.
Figure reproduced with permission from
[51].
Cross-linking of
interacting loci
Restriction digestion
Fragmentation
Biotin labeling of ends
DNA shearing
*
*
*
*
Immunoprecipitation
*
Ligation
*
DNA shearing
*
*
DNA purification
Inverse PCR
PCR
Multiplexed LMA
DNA
sequencing
DNA
sequencing
Ligation product
Detection
ChIA-PET
4C
3C
5C
Hi-C
