Biology Reference
In-Depth Information
(A)
(B)
Dam
scaffold protein
express Dam-fusion protein
in cells or tissue
crosslink;
isolate and fragment
chromatin
DNA in contact
with scaffold becomes
adenine-methylated
isolate genomic DNA
immuno-purify
with antibody against
scaffold protein
+
selectively amplify
adenine-methylated
DNA fragments
de-crosslink;
purify DNA
sequencing or
genomic tiling array
sequencing or
genomic tiling array
FIGURE 7.2
DamID and ChIP methods to map genome
e
scaffold interactions.
tethered Dam, and as a consequence are tagged by adenine
methylation (
m6
A). Because this covalent modification
does not occur endogenously, contacts with the scaffold can
be inferred from the
m6
A pattern, which can be mapped
genome-wide using methods that employ methylation-
sensitive restriction enzymes combined with a readout
based on genomic tiling arrays or high-throughput
sequencing.
Both methods yield very similar datasets that provide
genome-wide views of the interactions of the genome with
the scaffold protein of interest. It is important to note that
neither method can discriminate direct protein
that a detected interaction occurs in only a fraction of the
cells at any time. Nevertheless, the application of ChIP and
DamID has yielded some remarkable insights into the
interactions of the genome with components of the major
scaffold, the nuclear envelope.
Nuclear Lamina
Genome Interactions
e
in Metazoans
The inner nuclear membrane of virtually all metazoan cells
is coated by the nuclear lamina (NL), a dense web of
protein fibers. This network is primarily made of special-
ized intermediate filament proteins named lamins, and
contains in addition a variety of other proteins, some of
which are transmembrane proteins that are inserted into the
inner nuclear membrane.
DNA
contacts from indirect contacts (i.e., via another protein).
Both methods currently require approximately 10 000 cells
or more, hence the resulting maps represent population
averages that must be interpreted accordingly: it is possible
e
