Biology Reference
In-Depth Information
a strain containing a deletion of
900 bp at the N terminus of
ino80
possessed
roughly a sixfold reduction in the phospho-S129 H2A ChIP signal near the
break 2 h after induction, indicating that this mutant does not behave identi-
cally to the
ino80
deletion.
111
Two hours is a relatively long time after break
induction, and there could be subtle delays in accumulation of phosphorylated
H2A S129 in some
ino80
mutants. However, this could also be partly due to
delays in DSB formation that have been observed in some mutant strains.
107
Purification of the INO80 complex from cells treated with MMS resulted in
co-immunoprecipitation of phosphorylated H2A.
109
This interaction was unaf-
fected when Arp5 or Arp8, Arp4, and actin were absent from the complex, but
was reduced when
nhp10
was deleted (although affinity for bulk histones was
slightly reduced too). The INO80 complex from the
nhp10
deletion strain also
lacked Ies3 and notably the recruitment of this mutant complex to a DSB was
defective
in vivo
.
109
Furthermore, enrichment of Ino80 was found to be
reduced in a strain in which H2A could not be phosphorylated
101
and in an
mec1/tel1/sml1
deletion strain.
109
In addition, myc-tagged Ino80 (and Arp4)
was pulled down from cell extracts by a phosphorylated S129 H2A peptide.
108
Together, these data demonstrate that Ino80 is recruited to chromatin contain-
ing phospho-S129 H2A adjacent to a DSB and both Nhp10/Ies3 and Arp4 have
been implicated in contributing to this interaction.
3. C
HROMATIN
R
EMODELING AT A
DSB
Like RSC, INO80 recruitment to a DSB has been linked to changes in
chromatin close to the break. However, these remodeling events occur much
later than the nucleosome mobilization catalyzed by the RSC complex. The first
report connecting INO80 to chromatin remodeling at a break observed a
decrease in the ChIP signal of H2B and H3 near the break relative to that
before the break was formed.
110
The authors interpreted this as eviction of
histones and found that eviction was reduced, but not eliminated in
arp8
and
mre11
deletion strains, while it was largely unaffected in a strain in which H2A
could not be phosphorylated. In their study, resection was found to be unaf-
fected in an
arp8
strain and this led them to conclude that INO80 remodeling
promotes loss of histones from the region containing phosphorylated H2A in
both an MRX-dependent manner and a resection-independent manner. How-
ever, it has since been reported that the loss of histones observed at a break is
tightly coupled to resection.
112
When the H3 ChIP signal was normalized to
input DNA at each time point, that is, accounting for loss of DNA to amplify
due to resection, it was discovered that there was no change in the histone to
DNA ratio.
112
This is in agreement with what had previously been found for
H2B.
21
These more recent studies would suggest that INO80 is not required to
evict histones from chromatin flanking a DNA DSB (but the histones are
instead lost as a consequence of resection), which is consistent with the fact
