Biology Reference
In-Depth Information
Rsc7 are important in establishing normal chromatin structure at
MATa
in the
absence of breaks, which demonstrates that RSC is present and functions at
this locus under normal growth conditions.
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3. N
UCLEOSOME
R
EMODELING AT A
DSB
Upon break induction, a change in chromatin structure occurs immediately
adjacent to the break. Nucleosome positions in chromatin extracted from
MNase-treated permeabilized yeast were visualized by indirect end labeling.
This assay showed repositioning of six nucleosomes away from the break on the
proximal side and a small increase in MNase cleavage immediately distal to the
break.
74,76
Consistent with mobilization of nucleosomes in the locality of a
break, chromatin isolated from cross-linked nuclei and then subjected to
MNase digestion was more efficiently cleaved after DSB induction when
analyzed by qPCR.
74
Increased efficiency of restriction enzyme cleavage at
sites close to the break is also consistent with enhanced accessibility of this
region after break induction.
74
The change in chromatin structure occurred
within 30 min, was Mre-11-independent, and occurred in G1, demonstrating
that it is an event distinct from subsequent MRX-dependent histone eviction.
Nucleosome mobilization on break formation is not unique to the
MAT
locus;
it was also shown to occur at an HO break introduced at the
URA3
and
LEU2
loci.
76
DSB-dependent nucleosome repositioning was found to be dependent on
the RSC complex but not on other chromatin-remodeling complexes such as
SWI/SNF, INO80, or Rad54.
76
Chromatin in a strain where Sth1 was repressed
by the addition of doxycycline did not undergo a dramatic change in MNase
sensitivity following break induction.
74
An
rsc2
strain also failed to show
increased MNase sensitivity to the same extent as the wild type (WT) following
break induction. In contrast, indirect end-labeling analysis established that
nucleosome repositioning still occurred in an
rsc2
strain (and
rsc7
and
rsc30
)
(although as mentioned earlier, the chromatin structure before HO cleavage
was altered in
rsc2
and
rsc7
compared to the WT). However, in this assay, a
defect in remodeling in an
rsc1
strain was detected.
76
The apparent discrep-
ancy in the
RSC1
versus
RSC2
dependency of the chromatin remodeling could
be explained by differences in the assays used. While the qPCR-based assay
analyzing MNase susceptibility may be more sensitive to changes in occupancy
within a population or ''breathing'' of nucleosomes, the indirect labeling assay
may be more sensitive to positional changes, and consequently both isoforms of
the RSC complex may be involved in the remodeling event. It should also be
remembered that by definition
rsc1
and
rsc2
strains retain some RSC function.
The relative contribution of the two isoforms could be dependent on cell cycle,
chromatin context, or genomic location.
