The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair.

Abstract

The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage.

Full text

Page 1

JCB: Article
The Rockefeller University Press $30.00
J. Cell Biol. Vol. 191 No. 1 31–43
www.jcb.org/cgi/doi/10.1083/jcb.201001160
JCB31
Correspondence to Brendan D. Price: brendan_price@dfci.harvard.edu
Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; DSB,
DNA double-strand break; IR, ionizing radiation; MEF, mouse embryo fibro-
blast; TSA, trichostatin A; ZFN, zinc finger nuclease.
Introduction
DNA double-strand break (DSB) repair involves the deposition
of DNA repair proteins onto the chromatin. Early events in the
DNA damage response include the rapid recruitment of the ATM
kinase to DSBs, followed by the phosphorylation of H2AX
(-H2AX) on chromatin domains that extend for hundreds of
kilobases on either side of the DSB (Bonner et al., 2008). The
mdc1 scaffold protein then binds to -H2AX (Stucki et al., 2005),
providing a docking platform for the deposition of DNA repair
proteins onto the chromatin at DSBs (Melander et al., 2008;
Spycher et al., 2008). Consequently, DSB repair can be nega-
tively impacted by local chromatin architecture, which may
restrict the ability of the DNA repair machinery to access and
repair DSBs. Mammalian cells use multiple chromatin remod-
eling pathways to alter chromatin structure during DSB repair.
For example, chromatin is hypersensitive to nuclease digestion
after DNA damage (Smerdon et al., 1978; Carrier et al., 1999;
Rubbi and Milner, 2003; Ziv et al., 2006), consistent with de-
compaction of a significant fraction of the chromatin in response
to DSBs. This global decompaction requires the ATM-dependent
phosphorylation of the kap1 heterochromatin-binding protein
(Ziv et al., 2006), indicating that phosphorylation of kap1 may
be essential for unpacking heterochromatin during DSB repair
(Goodarzi et al., 2008). In addition to global effects on chromatin
structure, biophysical studies indicate that there is a localized
expansion of the chromatin adjacent to DSBs (Kruhlak et al.,
2006), a process which was independent of the ATM kinase.
These distinct pathways of chromatin remodeling indicate that
cells use multiple mechanisms to create the open, accessible
regions, which are critical for DSB repair.
Chromatin remodeling complexes contain SWI/SNF-
related DNA-dependent ATPases, and use the energy of ATP
hydrolysis to alter histone–DNA interactions, to reposition nucleo-
somes along the DNA (nucleosome sliding) or insert histone
variants (Cairns, 2005). In yeast, the INO80, Swr1, and NuA4
complexes are recruited to enzymatically induced DSBs (Downs
et al., 2004; van Attikum et al., 2004; Papamichos-Chronakis
et al., 2006) and are required for DSB repair (van Attikum et al.,
2004; Tsukuda et al., 2005). However, although mammalian
T
breaks (DSBs). Consequently, remodeling of the chromatin
landscape adjacent to DSBs is vital for efficient DNA
repair. Here, we demonstrate that DNA damage destabi-
lizes nucleosomes within chromatin regions that corre-
spond to the -H2AX domains surrounding DSBs. This
nucleosome destabilization is an active process requiring
the ATPase activity of the p400 SWI/SNF ATPase and
histone acetylation by the Tip60 acetyltransferase. p400
he complexity of chromatin architecture presents a
significant barrier to the ability of the DNA repair
machinery to access and repair DNA double-strand
is recruited to DSBs by a mechanism that is independent
of ATM but requires mdc1. Further, the destabilization of
nucleosomes by p400 is required for the RNF8-dependent
ubiquitination of chromatin, and for the subsequent re-
cruitment of brca1 and 53BP1 to DSBs. These results iden-
tify p400 as a novel DNA damage response protein and
demonstrate that p400-mediated alterations in nucleo-
some and chromatin structure promote both chromatin
ubiquitination and the accumulation of brca1 and 53BP1
at sites of DNA damage.
The p400 ATPase regulates nucleosome stability
and chromatin ubiquitination during DNA repair
Ye Xu,1 Yingli Sun,1 Xiaofeng Jiang,1 Marina K. Ayrapetov,1 Patryk Moskwa,1 Shenghong Yang,1 David M. Weinstock,2
and Brendan D. Price1
1Division of Genomic Stability and DNA Repair, Department of Radiation Oncology, and 2Department of Medical Oncology, Dana-Farber Cancer Institute,
Harvard Medical School, Boston, MA 02115
© 2010 Xu et al. This article is distributed under the terms of an Attribution–Noncommercial–
Share Alike–No Mirror Sites license for the first six months after the publication date (see
http://www.rupress.org/terms). After six months it is available under a Creative Commons
License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at
http://creativecommons.org/licenses/by-nc-sa/3.0/).
T H E J O U R N A L O F C E L L B I O L O G Y

Page 2

JCB • VOLUME 191 • NUMBER 1 • 2010 32
from bleomycin-treated cells. In Fig. 1 b, nuclei were first ex-
tracted with 1.0 M NaCl (Fig. 1 b, lanes 2 and 5; NaCl fraction),
the nuclei collected by centrifugation, and the histones remain-
ing in the nuclear pellet extracted by acid extraction (Fig. 1 b,
lanes 3 and 6; Pellet). For comparison, total histones (obtained
by acid extraction) are shown (Fig. 1 b, lanes 1 and 4; Total).
Control cells contained low but detectable levels of -H2AX
(lane 1). No detectable -H2AX, H2AX, or H3 was seen after
fractionation of control cells in 1.0 M NaCl (Fig. 1 b, lane 2), in-
dicating that this pool of -H2AX remains associated with the
chromatin (lane 3). In contrast, bleomycin treatment led to the
elution of low but detectable levels of histones H2AX and H3
(compare lanes 4 and 5). -H2AX levels were increased by bleo-
mycin (compare lanes 1 and 4), and a significant fraction of this
-H2AX was released by NaCl fractionation (lane 5). Impor-
tantly, although only a small fraction of the total H2AX was eluted
cells exhibit alterations in chromatin structure during DSB repair
(Rubbi and Milner, 2003; Kruhlak et al., 2006; Ziv et al., 2006),
the mechanistic aspects of this process, including the protein
complexes involved, the nature of the changes in chromatin
structure, and how chromatin structure impacts DSB repair, are
poorly understood. To gain insight into how chromatin remodel-
ing contributes to DSB repair in mammalian cells, we examined
the role of the p400 chromatin remodeler in regulating the stabil-
ity of nucleosomes adjacent to DSBs. p400 is a SWI/SNF DNA-
dependent ATPase (Chan et al., 2005) that functions to alter
DNA–histone interactions and facilitates the insertion of histone
variants, including H2A.Z, into gene promoters (Gévry et al.,
2007). p400 is a component of the mammalian NuA4 complex
(Downs et al., 2004; Doyon et al., 2004), and is associated with
the Tip60 acetyltransferase. Tip60 is required for DSB repair
(Bird et al., 2002; Sun et al., 2005, 2007, 2009; Murr et al., 2006;
Gorrini et al., 2007), and can acetylate histones after DNA dam-
age (Bird et al., 2002; Kusch et al., 2004; Murr et al., 2006; Jha
et al., 2008). Further, p400 and Tip60 function in a common
pathway to regulate apoptotic responses to DNA damage
(Mattera et al., 2009), implying that the p400 ATPase and the
Tip60 acetyltransferase may function to alter chromatin structure
during DSB repair. Here, using DSBs generated by both geno-
toxic agents and by designer zinc finger nucleases to target a
specific endogenous locus, we describe a new role for p400 in
altering nucleosome stability during DSB repair.
Results
Histone–DNA interactions are very stable, and histones are
only released from chromatin by NaCl concentrations in excess
of 1.5 M (von Holt et al., 1989; Kimura and Cook, 2001; Shechter
et al., 2007). If the stability of the histone–DNA interaction is
reduced by DNA damage, it should be possible to preferentially
elute histones from damaged chromatin by biochemical frac-
tionation. To test this, cells were exposed to the radiomimetic
agent bleomycin to introduce DSBs, and isolated nuclei were
fractionated with increasing concentrations of NaCl and the
histones in each fraction monitored (Fig. 1 a). Increasing the
NaCl concentration from 0.2 to 0.6 M did not elute detect-
able amounts of histone from the chromatin. However, histones
H2AX, -H2AX, H2B, H3, and H4 were released from bleo-
mycin treated cells at 0.8–1.0 M NaCl, but not from untreated
cells. At concentrations of NaCl of 1.5 M and above, disruption
of histone–DNA interactions occurs (von Holt et al., 1989), as
seen by release of histones from untreated cells at this concen-
tration of NaCl (Fig. 1 a). This indicates that, after DNA dam-
age, the interaction between histones and DNA in the chromatin
is reduced, allowing histones to be extracted from the chromatin
at lower levels of NaCl.
A key question is to determine the source of the fraction-
ated histones. Changes in chromatin structure after DNA dam-
age may be localized to regions adjacent to DSBs (Kruhlak et al.,
2006) or associated with relaxation of the entire chromatin struc-
ture (Ziv et al., 2006). If the histones released by NaCl fractionation
originate from DSBs, then, because the majority of -H2AX
occurs at DSBs, the method should preferentially release -H2AX
Figure 1. DNA damage reduces the interaction between DNA and his-
tones. (a) 293T cells were exposed to 5 µM bleomycin for 30 min. Isolated
nuclei were extracted in buffer containing the indicated concentration of
NaCl. -H2AX, H2AX, H2B, H3, and H4 detected by Western blot. Mem-
branes were stained with ponceau S to ensure equal loading. (b) 293T
cells were untreated (control) or exposed to 5 µM bleomycin for 30 min.
Total chromatin-associated histones were obtained by acid extraction.
In parallel, cells were first extracted with 1.0 M NaCl and the nuclei col-
lected by centrifugation. The histones remaining in the nuclear pellet were
then purified by acid extraction. -H2AX, H2AX, and H3 were detected
by Western blot. Table: Relative enrichment of -H2AX in the Total and
NaCl extracts was calculated by normalizing -H2AX scan intensities to
H2AX signal in each extract, expressed as [-H2AXTotal]/[H2AXTotal] and
[-H2AXNaCl]/[H2AXNaCl]. Results from three independent replicates, ± SD.
(c) 293T cells were treated with 5 µM bleomycin and extracted in 1.0 M
NaCl. -H2AX, H2AX, H2B, H3, and H4 were detected by Western blot.
Equal loading confirmed by ponceau S staining.

Page 3

33p400 regulates chromatin ubiquitination • Xu et al.
(see Fig. 6 c), no histones were eluted from bleomycin-treated
cells by NaCl treatment (Fig. 3 a), indicating that p400 is required
to reduce nucleosome stability after DNA damage. If p400
functions as part of the NuA4 complex, loss of the Trrap sub-
unit, which is thought to function as a scaffold protein for the
NuA4 complex, should also impact nucleosomal remodeling.
Indeed, when Trrap levels were reduced with shRNA (Fig. S2 b),
the ability of NaCl fractionation to release histones from the
chromatin was lost (Fig. 3 b), demonstrating that Trrap is re-
quired for reducing nucleosome stability. Chromatin remodel-
ing is facilitated by acetylation of histones, which can reduce the
stability of histone–DNA interaction and inhibit the packing of
nucleosome arrays (Brower-Toland et al., 2005; Dion et al., 2005;
by NaCl after bleomycin treatment, this eluted fraction was sig-
nificantly enriched for -H2AX compared with the total (Fig. 1 b,
lane 5). To determine the exact enrichment of -H2AX, Western
blots were quantitated (Fig. S1 a), and the levels of -H2AX
normalized to the level of H2AX in each extract. NaCl-extracted
histones contained 3.2-fold more -H2AX compared with the
overall amount in the total chromatin (Fig. 1 b, table). The his-
tones extracted from the chromatin by NaCl after DNA damage
are therefore enriched for -H2AX, indicating that they prefer-
entially originate from regions of the chromatin containing high
levels of -H2AX—that is, from chromatin regions adjacent to
the DSBs. However, we cannot exclude the possibility that a frac-
tion of the released histones are derived from chromatin domains
that are not associated with the DSB.
Next, we monitored the time course of changes in histone–
DNA interaction after DNA damage. Histone sensitivity to
NaCl fractionation was detected 15–30 min after bleomycin
addition (Fig. 1 c), and was maintained for at least 4 h during
continuous bleomycin exposure. In contrast, acute exposure to
ionizing radiation (IR) induced a more limited chromatin response,
such that the chromatin-associated histones were only sensitive
to NaCl fractionation for 30 min after irradiation (Fig. 2 a).
The destabilization of the chromatin in response to acute DNA
damage (IR) is therefore an early but transient response to DSBs.
The decrease in the stability of histone–DNA interactions
was only detected 15 min after DSB production (Fig. 1 c and 2 a),
whereas ATM activation and phosphorylation of H2AX were
maximal within 5 min of exposure to IR (Fig. 2 b). Because the
altered DNA–histone stability occurs after ATM activation, we
determined if it was regulated by the ATM kinase. Both A-T
cells (which lack ATM protein) and A-T cells complemented with
ATM (A-TATM; Sun et al., 2005, 2009) had similar sensitivity to
NaCl extraction after exposure to bleomycin (Fig. 2 c). In addi-
tion, neither inhibition of ATM kinase by Ku55933 (Hickson
et al., 2004; Fig. 2 d and Fig. S1 b) nor inactivation of the MRN
complex (Fig. S1), which regulates ATM (Lee and Paull, 2005),
blocked the reduced histone–DNA interaction seen after DSB
production (Fig. S1 d). Previous work demonstrated that global
chromatin relaxation required phosphorylation of kap-1 by ATM
(Ziv et al., 2006). However, the reduced stability of DNA–histone
interactions seen after DNA damage was independent of both
ATM and kap1 phosphorylation (Fig. S1 b). The localized changes
in chromatin structure reported here are therefore mechanisti-
cally distinct from the changes in chromatin structure mediated
by the ATM-kap1 pathway (Ziv et al., 2006). The results are
consistent with a model in which, immediately after DNA dam-
age, the chromatin structure at or near DSBs is altered, weaken-
ing the interaction between DNA and histones, and therefore
allowing histones to be released from the chromatin in 1.0 M
NaCl. We refer to this reduction in histone–DNA interaction
after DNA damage as reduced nucleosome stability.
The Tip60 (Gorrini et al., 2007; Sun et al., 2009) and Trrap
(Murr et al., 2006; Robert et al., 2006) subunits of the NuA4 com-
plex have been implicated in DSB repair (Downs et al., 2004;
Doyon et al., 2004). Here, we examined if the p400 SWI/SNF
ATPase subunit of NuA4 was required for the decrease in nucleo-
some stability at DSBs. When p400 was reduced by shRNA
Figure 2. ATM is not required for altered nucleosome stability after DNA
damage. (a) 293T cells were irradiated (10Gy), fractionated in 1.0 M
NaCl, and released histones detected by Western blot. (b) 293T cells
were irradiated (10Gy) and whole-cell extracts examined by Western
blot for total -H2AX, H2AX, ATM, and pATM. (c) GM5849 A-T cells
complemented with either vector (A-T) or full-length ATM (A-TATM) were
exposed to 5 µM bleomycin, fractionated in 1.0 M NaCl, and released
histones detected by Western blot. (d) 293T cells were incubated with 10 µM
of the ATM kinase inhibitor Ku55933 for 60 min, followed by 5 µM
bleomycin. Cells were fractionated in 1.0 M NaCl, and released histones
detected by Western blot.

Page 4

JCB • VOLUME 191 • NUMBER 1 • 2010 34
effects are due to loss of p400 or loss of the entire NuA4 com-
plex. To distinguish between these possibilities, p400’s ATPase
activity was inactivated by mutagenesis (Samuelson et al., 2005).
Cells expressing wild-type p400 (Fig. S2 a) showed decreased
nucleosome stability after bleomycin treatment, whereas this
effect was lost in cells expressing the p400ATPase mutant (Fig. 3 d).
Similar results were seen after IR exposure (Fig. S2 a). In addi-
tion, preincubation of the p400ATPase cells with TSA to induce
histone hyperacetylation did not restore the ability of bleomycin
to decrease nucleosome stability (Fig. 3 d). Histone acetylation,
on its own, is therefore insufficient to decrease nucleosome
stability at DSBs. Fig. 3 demonstrates that both Tip60’s acetyl-
transferase activity and p400’s ATPase activity are required for
regulating nucleosome stability at DSBs. Further, because p400,
Tip60, and Trrap are components of the mammalian NuA4 com-
plex (Doyon et al., 2004), it is likely that these proteins regulate
DSB repair as part of the NuA4 complex.
Next, the role of p400 in the DNA damage response was
investigated. Fig. 4 a demonstrates that p400ATPase cells are sig-
nificantly more sensitive to ionizing radiation than cells express-
ing wild-type p400. Cells expressing the p400ATPase also exhibited
Shogren-Knaak et al., 2006; Choi and Howe, 2009). The Tip60
acetyltransferase associates with NuA4 and acetylates H4 after
DNA damage (Kusch et al., 2004; Murr et al., 2006; Jha et al.,
2008), suggesting that Tip60 may regulate nucleosome stability.
Cells expressing a previously described catalytically inactive
version of Tip60 (Tip60HD; Fig. S2c; Sun et al., 2005) lacked
any detectable decrease in nucleosome stability after DNA damage
(Fig. 3 c), indicating that Tip60’s acetyltransferase activity is re-
quired for alteration of nucleosome stability after DNA damage.
To confirm that histone acetylation is crucial, Tip60HD cells were
pretreated with the histone deacetylase inhibitor trichostatin A
(TSA). TSA increased H4 acetylation levels (Fig. S2 d) and re-
stored the ability of bleomycin to decrease nucleosome stability
in the Tip60HD cells (Fig. 3 c). Hyperacetylation of histones
through histone deacetylase inhibition therefore compensates
for the loss of Tip60’s acetyltransferase activity, demonstrating
that histone acetylation by Tip60 is required for reduced nucleo-
somal stability at DSBs.
p400 and Trrap are integral components of NuA4 (Doyon
et al., 2004). Reducing p400 or Trrap with shRNA may dis-
assemble NuA4, making it difficult to determine if the observed
Figure 3. Tip60, p400, and Trrap are required for altered nucleosome stability. (a) 293T cells expressing a nonspecific shRNA (vector) or shRNA targeting
p400 were exposed to 5 µM bleomycin. Cells were fractionated in 1.0 M NaCl, and released histones detected by Western blot. (b) HeLa cells express-
ing vector or shRNA targeting Trrap were exposed to bleomycin. Cells were fractionated in 1.0 M NaCl, and released histones detected by Western blot.
(c) 293T cells expressing either HA-Tip60 or catalytically inactive HA-Tip60HD were incubated with 5 µM bleomycin. Cells were fractionated in 1.0 M
NaCl, and released histones detected by Western blot. Where indicated, cells were pretreated with 300 nM TSA for 2 h. (d) 293T cells expressing either
HA-p400 or HA-p400ATPase were incubated with 5 µM bleomycin. Cells were fractionated in 1.0 M NaCl, and released histones detected by Western blot.
Where indicated, cells were pretreated with 300 nM TSA for 2 h.

Page 5

35p400 regulates chromatin ubiquitination • Xu et al.
using the ZFN ChIP approach. p400 and p400ATPase cells were
transfected with the ZFN nuclease to create DSBs, and then
extracted in 1.0 M NaCl to remove histones from regions of de-
creased nucleosome stability. ChIP was then performed using an
anti-H3 antibody to look for depletion of H3 on the chromatin
adjacent to the ZFN site on chromosome 19. For comparison, we
examined a randomly selected region on chromosome 6. Histone
H3 was specifically removed from the chromatin adjacent to the
ZFN DSB by NaCl extraction (1.5 kb; Fig. 5 d, Chr19) in
wild-type cells, but not in p400ATPase cells. In contrast, no signifi-
cant release of H3 was detected at an unrelated region on chromo-
some 6 (Fig. 5 d, Chr 6). This is consistent with the results in Fig. 1 b,
and supports the proposal that the decrease in nucleosome stabil-
ity is preferentially localized to chromatin domains adjacent to
the DSB on chromosome 19.
One potential explanation for the inability of the p400ATPase
protein to alter nucleosome stability is that it is not recruited to
DSBs. However, Fig. 6 a demonstrates that both p400 and the
p400ATPase proteins were recruited to DSBs with similar effi-
ciency. In addition, p400 was recruited to DSBs in Tip60HD cells
(Fig. 6 a). Thus, the inability of the p400ATPase and Tip60HD mutants
to decrease nucleosome stability (Fig. 3) is not due to the failure
to recruit these proteins to DSBs, but to the lack of intrinsic cat-
alytic activity. Tip60 acetylates histone H4 after DNA damage
(Bird et al., 2002; Murr et al., 2006), suggesting that Tip60 may
regulate nucleosome stability through acetylation of histone
H4. ChIP analysis confirmed that H4 acetylation was increased
in cells with ZFN-induced DSBs, but not in cells expressing
the catalytically inactive Tip60HD (Fig. 6 b). We also examined
if the ability of Tip60 to acetylate histones at DSBs requires
p400’s ATPase activity. Fig. 6 b demonstrates that, although his-
tone H4 was acetylated after DSB generation in wild-type p400
cells, H4 acetylation was slightly reduced in cells expressing
the p400ATPase mutant. Because p400 and Tip60 are part of the
NuA4 complex, it is possible that the p400ATPase mutant affects
the intrinsic catalytic activity of Tip60. Fig. 6 c (inset) demon-
strates that p400 and Tip60 are specifically coprecipitated from
cells, as previously shown (Doyon et al., 2004), and that the
p400–Tip60 interaction was not altered by DNA damage (Fig. 6 c).
Next, p400 was immunopurified and the associated acetyltrans-
ferase activity measured as we previously described (Sun et al.,
2005). The acetyltransferase activity associated with immuno-
purified p400 was increased by DNA damage (Fig. 6 c); cells
expressing shRNA to p400 or immunopurification with IgG ex-
hibited little or no associated acetyltransferase activity or Tip60.
Significantly, the catalytically inactive p400ATPase had similar
levels of basal and DNA damage–activated acetyltransferase
activity as the wild-type p400 (Fig. 6 c). The p400ATPase mutant
is therefore recruited to DSBs (Fig. 6 a) and the Tip60 associ-
ated with the p400ATPase mutant remains responsive to DNA
damage (Fig. 6 c). However, the levels of histone H4 acetylation
were slightly reduced in cells expressing the p400ATPase mutant
(Fig. 6 b), suggesting that p400’s ATPase activity is required for
efficient acetylation of histone H4 at DSBs. This could occur,
for example, through p400-mediated changes in chromatin
structure that facilitate Tip60 acetylation of histone H4. In con-
clusion, Fig. 6 demonstrates that recruitment of p400 to DSBs
a more rapid exit from G2 arrest than control cells (Fig. S2 e),
consistent with a small defect in G2 arrest in these cells. In addi-
tion, irradiation of the p400ATPase cells resulted in significantly
higher levels of chromosome aberrations compared with cells
expressing wild-type p400 (Fig. 4 b). These observations are
consistent with a significant DNA repair defect in the p400ATPase
cells, and define p400 as a novel DNA damage response protein
that is required for cells to survive exposure to IR.
Although many DNA damage responses form foci at
DSBs, we were unable to detect p400 foci using immunofluores-
cent approaches. Accordingly, we used an alternative, chromatin
immunoprecipitation (ChIP)–based approach to monitor recruit-
ment of p400 to DSBs. Zinc finger nucleases (ZFN) are custom-
engineered nucleases in which the catalytic domain of the Fok1
endonuclease is linked to an engineered zinc finger protein, cre-
ating sequence-specific nucleases that generate a DSB at unique
chromatin sites (Urnov et al., 2005). DSBs caused by ZFNs at
endogenous loci in human cells can be used to insert or alter ge-
nomic sequences through homology-directed repair with donor
DNA (Urnov et al., 2005; Hockemeyer et al., 2009), and have
been used to examine chromosomal translocations (Brunet et al.,
2009). The AAVS1 ZFN (p84-ZFN) introduces a single DSB into
intron 1 of the PPP1R12C gene on chromosome 19 (Hockemeyer
et al., 2009). p84-ZFN was used to introduce a single DSB into
chromosome 19, and ChIP analysis was then used to monitor
p400 recruitment to this unique DSB. Transient transfection of
the p84-ZFN generates DSBs in 40–50% of cells into which it is
introduced (Fig. 5 a and Fig. S3 b). The ZFN-generated DSB in-
creases the levels of -H2AX (Fig. S3 a), and ChIP analysis
demonstrates the formation of -H2AX domains extending at
least 3.5 kb on either side of the ZFN DSB (Fig. 5 b). Signifi-
cantly, ChIP analysis demonstrated that p400 was also recruited
to chromatin domains adjacent to the DSB (Fig. 5 c), indicating
that p400, like other DNA damage response proteins, is actively
recruited to and retained at DSBs. Next, we examined if the de-
creased nucleosome stability mediated by p400 could be detected
Figure 4. Loss of p400’s ATPase activity increases radiosensitivity and
chromosome aberrations. (a) HeLaS3 cells expressing either p400 (s) or
p400ATPase () were irradiated, and the number of surviving colonies mea-
sured 12 d later. Each data point represents the average of three indepen-
dent assays, results ± SD. (b) p400 or p400ATPase cells were untreated (0)
or irradiated (2Gy) and allowed to recover for 14 h. Metaphase spreads
were subsequently scored visually for chromosome aberrations. Results ex-
pressed as aberrations per cell (n = 50 cells), and P values determined
using t test.

Page 6

JCB • VOLUME 191 • NUMBER 1 • 2010 36
of H2AX is therefore required for p400-dependent alterations in
nucleosome stability.
-H2AX recruits mdc1 to DSBs, which then serves as a
platform to concentrate DNA repair proteins at sites of DNA dam-
age (Spycher et al., 2008). To determine if mdc1 was required to
recruit p400 to DSBs, mdc1 was silenced by shRNA (Fig. S4,
c and d). Silencing of mdc1 blocked the p400-dependent de-
crease in nucleosome stability (Fig. 7 b). Similar results were
seen with mdc1/ MEFs (Fig. S4 e). These results imply that re-
cruitment of mdc1 to -H2AX is required to position p400 at
DSBs. Immunoprecipitation of p400 demonstrated that p400
and mdc1 were present as a preformed complex and that the
p400–mdc1 complex was not altered by DNA damage (Fig. S4 f),
consistent with reports that mdc1 forms stable complexes with
several DNA damage response proteins (Melander et al., 2008;
Spycher et al., 2008). In addition, ChIP assays (Fig. 7 c) demon-
strate that p400 is not recruited to ZFN-generated DSBs in the ab-
sence of mdc1. Together, the immunoprecipitation (Fig. S4 f)
and ChIP data (Fig. 7 c) are consistent with a model in which
p400 is recruited to DSBs through interaction between mdc1
and -H2AX. The p400-dependent decrease in nucleosome
stability therefore occurs after phosphorylation of H2AX and
assembly of mdc1 onto the chromatin.
DNA damage leads to increased ubiquitination of the
chromatin by the RNF8 ubiquitin ligase (Huen et al., 2007; Kolas
et al., 2007; Mailand et al., 2007). To determine if this ubiqui-
tination contributes to decreased nucleosome stability, RNF8
was targeted using both siRNA (Fig. S4 h) and by overexpress-
ing an HA-RNF8 protein lacking the ring domain (Huen et al.,
2007; Fig. 7 d and Fig. S4 g). In both cases (Fig. 7 d and Fig. S4 h),
is independent of the ATPase activity of p400, indicating that
the failure to detect decreased nucleosome stability in p400ATPase
cells is due to the loss of the intrinsic ATPase activity of p400.
Tip60 can also acetylate lysine 5 of H2AX (Jha et al.,
2008), and may regulate the turnover of the Drosophila H2A.Z
homologue, H2Av, at DSBs (Kusch et al., 2004). However, mu-
tation of lysine 5, or all four potential lysine acetylation sites in
the N-terminus of H2AX (Fig. S4 a), had minimal impact on the
p400-dependent decrease in nucleosome stability. Acetylation
of histones by Tip60 may therefore have distinct functions, with
acetylation of H4 mediating the altered nucleosome stability
observed here, and acetylation of H2AX contributing to turn-
over of -H2AX (Kusch et al., 2004; Ikura et al., 2007).
-H2AX plays a key role in recruiting DNA damage re-
sponse proteins at DSBs, suggesting that -H2AX may recruit
p400 to DSB. MEFs derived from H2AX/ mice expressing
either vector (H2AX/) or complemented with wild-type H2AX
(H2AXwt) were exposed to bleomycin (Fig. 7 a). H2AXwt cells
exhibited decreased nucleosome stability after bleomycin ex-
posure (Fig. 7 a), whereas H2AX/ cells displayed no significant
decrease in nucleosome stability. Although -H2AX is required
to decrease nucleosome stability, ATM, which directly phosphory-
lates H2AX, was not required (Fig. 1). This is due to the fact
that H2AX can be phosphorylated by both the ATM and DNA-
PKcs kinases, so that loss of ATM activity does not significantly
impair the formation of -H2AX domains (Stiff et al., 2004).
In fact, when H2AXwt MEFs were treated with the dual specificity
ATM/DNA-PKcs inhibitor wortmannin (which inhibits phosphory-
lation of H2AX; Fig. S4 b; Stiff et al., 2004), the decrease in nucleo-
some stability caused by bleomycin was inhibited. Phosphorylation
Figure 5. p400 is recruited to DSBs gener-
ated by the p84-ZFN. (a) 293T cells were
transiently transfected with p84-ZFN, DNA
extracted and examined by qPCR to monitor
the fraction of cells with DSBs. (b) 293T cells
were transiently transfected with vector () or
p84-ZFN (+). 18 h later, cells were processed
for ChIP analysis using anti–-H2AX antibody
and primer pairs located left and right of the
DSB. ChIP data points were calculated as IP
DNA/input DNA (see Materials and methods).
Fold enrichment is the relative fold increase in
signal from the ZFN samples compared with
the vector samples, which were normalized to
give a value of 1. Results ± SE (n = 3). (c) 293T
cells were transiently transfected with vector
() or p84-ZFN (+) as described in b. Cells
were processed for ChIP analysis using anti-
p400 antibody. Results ± SE (n = 3). (d) 293T
cells expressing p400 or p400ATPase were tran-
siently transfected with vector () or p84-ZFN (+).
18 h later, cells were preextracted in 1.0 M
NaCl to release histones, and the nuclear pel-
let collected and processed for ChIP analysis
as described in Materials and methods. Sam-
ples were immunoprecipitated with anti-H3
antibody. qPCR was performed using primers
located at +1.5 kb relative to the ZFN site on
chromosome 19 (Chr19), or at nucleotide po-
sitions 122,788,208 and 122,788,440 on
chromosome 6 (Chr 6). Results ± SE (n = 3).

Page 7

37 p400 regulates chromatin ubiquitination • Xu et al.
suppression of RNF8 did not affect the p400-dependent de-
crease in nucleosome stability, indicating that RNF8-dependent
ubiquitination of chromatin does not contribute to the p400-
dependent decrease in nucleosome stability.
Because p400 functions at a relatively late stage in the
DNA damage signaling process, we determined if the recruitment
of late components of the DNA damage response, including 53BP1
and brca1, required p400. Consistent with previous reports
(Huen et al., 2007; Mailand et al., 2007), recruitment of brca1 to
DSBs was not detected until 15–30 min after exposure to IR
(Fig. 8 a). Strikingly, no significant accumulation of brca1 was
detected in cells expressing the p400ATPase mutant (Fig. 8, a and c),
although formation of -H2AX foci was normal (Fig. 8 c). Sim-
ilarly, the recruitment of 53BP1 to DSBs was significantly impaired
at early times in p400ATPase cells, although 53BP1 eventually
accumulated to near-normal levels by 60 min in the p400ATPase
cells (Fig. 8 b). The recruitment of both brca1 and 53BP1 to
DSBs is therefore dependent on p400, and is consistent with
previous reports that another component of NuA4, Trrap, is re-
quired for brca1 and 53BP1 accumulation at DSBs (Murr et al.,
2006). The recruitment of brca1 to DSBs also requires recruitment
of the RNF8 ubiquitin ligase, which ubiquitinates chromatin-
associated proteins and facilitates the recruitment of RAP80
and the ccdc98–brca1 complex to DSBs (Huen et al., 2007;
Figure 6. Acetylation of histone H4 by the p400–Tip60 complex.
(a) 293T cells expressing wild-type Tip60 (Tip60wt), catalytically inactive
Tip60 (Tip60HD), wild-type p400 (p400), or p400 lacking ATPase activ-
ity (p400ATPase) were transiently transfected with vector () or p84-ZFN
(+), and ChIP assays using anti-p400 antibody performed using primers
located at +1.5 kb relative to the DSB. Results ± SE (n = 3). (b) 293T cells
expressing Tip60wt, Tip60HD, p400, or p400ATPase were transiently trans-
fected with vector () or p84-ZFN (+). 18 h later, ChIP assays were per-
formed using anti-acetylH4 antibody (AcH4) and primers located +1.5 kb
relative to the DSB. Results ± SE (n = 3). (c) p400 was immunopurified from
293T cells expressing either HA-p400, HA-p400ATPase, or cells in which
p400 was silenced with shRNA and the associated Tip60 acetyltransferase
activity measured. Cells were exposed to bleomycin (+: 5 µM for 40 min)
where indicated. Inset: Immunoprecipitates were separated by SDS-PAGE,
and p400 and coprecipitating Tip60 detected by Western blot.
Figure 7. Recruitment of p400 to DSBs requires mdc1. (a) H2AX/ MEFs
or H2AX/ MEFs complemented with H2AX (H2AXwt) were exposed to
5 µM bleomycin. 20 µM Wortmannin was added 60 min before bleo-
mycin. Cells were fractionated in 1.0 M NaCl, and released histones de-
tected by Western blot. (b) 293T cells expressing vector or mdc1 shRNA
(shmdc1) were exposed to 5 µM bleomycin. Cells were fractionated in 1.0 M
NaCl, and released histones detected by Western blot. (c) 293T cells ex-
pressing GFP or mdc1 shRNA were transiently transfected with vector or
p84-ZFN. 18 h later, ChIP assays using p400 antibody and primer pairs
located at +1.5 kb were performed. Results ± SE (n = 3). (d) 293T cells
stably expressing FLAG-RNF8 or RNF8 lacking the catalytic domain (FLAG-
RNF8ring), which functions as a dominant-negative inhibitor of endog-
enous RNF8 function (Huen et al., 2007), were exposed to bleomycin.
Cells were fractionated in 1.0 M NaCl, and released histones detected
by Western blot.

Page 8

JCB • VOLUME 191 • NUMBER 1 • 2010 38
antibody revealed extensive ubiquitination of a large chromatin
domain after DSB generation by the p84-ZFN (Fig. 9 c); forma-
tion of this domain was significantly impaired in cells express-
ing the p400ATPase mutant. Fig. 9 clearly demonstrates that p400
is required for the RNF8-dependent ubiquitination of chromatin
adjacent to DSBs. The failure to detect significant accumulation of
brca1 in cells expressing the p400ATPase mutant (Fig. 8 a) therefore
reflects the inability of the p400ATPase mutant cells to ubiqui-
tinate the chromatin, which is required to localize the rap80–
brca1 complex onto the chromatin (Huen et al., 2007; Kolas et al.,
2007; Mailand et al., 2007). This implies that p400-dependent
changes in nucleosome stability are required either to recruit
RNF8 to DSBs, or to facilitate the ubiquitination of chromatin
target proteins by RNF8 at DSBs. Fig. 9, d and e demonstrate
that RNF8 was efficiently recruited to DSBs in both normal and
p400ATPase cells, although the formation of FK2 foci was signifi-
cantly impaired in the p400ATPase cells. Overall, the results dem-
onstrate that p400’s ATPase activity is required to facilitate the
ubiquitination of chromatin target proteins by RNF8 adjacent to
DSBs. The failure to recruit brca1 to DSBs in p400ATPase cells
therefore reflects the failure of RNF8 to ubiquitinate the chro-
matin and to create binding sites for the rap80–brca1 complex.
Discussion
Here, we reveal that DSBs lead to the formation of chromatin
domains in which the histone–DNA interaction within nucleo-
somes is weakened. However, the nucleosomes remain an inte-
gral part of the chromatin, indicating that the decrease in
histone–DNA interaction reflects a switch in chromatin confor-
mation to a more open, flexible structure after DNA damage.
The p400-dependent decrease in nucleosome stability observed
here was preferentially located within the -H2AX domains that
form adjacent to DSBs (Bonner et al., 2008). This conclusion is
supported by several lines of evidence. First, the histones re-
leased from the chromatin by NaCl fractionation are enriched
for -H2AX relative to other histones, indicating that they are
preferentially derived from the -H2AX domains adjacent to
the DSB. Second, ChIP assays indicate that NaCl fractionation
removed histone H3 from regions adjacent to the ZFN-generated
DSBs. Third, both Tip60 and p400 are localized to the chromatin
adjacent to the DSB. Collectively, these results imply that the
p400-mediated decrease in nucleosome stability is preferen-
tially focused within chromatin domains contiguous with the DSB.
Previous work has shown that DNA damage can alter chromatin
architecture by two distinct pathways—an ATM-independent
pathway that alters local chromatin structure (Shroff et al.,
2004; Tsukuda et al., 2005; Kruhlak et al., 2006) and an ATM/
phospho-kap1–dependent pathway that initiates global chro-
matin relaxation, including heterochromatin (Ziv et al., 2006;
Goodarzi et al., 2008). However, the p400-mediated decrease in
nucleosome stability did not require either ATM or kap1 phosphory-
lation, indicating that it is distinct from the phospho-kap1/
ATM–mediated pathway of global decompaction previously
described (Ziv et al., 2006). Our results are therefore more con-
sistent with a previous report that chromatin structural changes
at DSBs are independent of ATM (Kruhlak et al., 2006). p400 is
Kolas et al., 2007; Mailand et al., 2007). To determine if p400
regulates brca1 recruitment by modulating RNF8 function, we
examined if p400 alters the ability of RNF8 to ubiquitinate its
target proteins. RNF8-mediated ubiquitination can be detected
using the FK2 antibody, which detects ubiquitin conjugated to
proteins (Huen et al., 2007; Kolas et al., 2007; Mailand et al.,
2007). FK2 foci were detected 15 min after exposure to IR, and
remained elevated for at least 60 min, whereas cells expressing
the p400ATPase mutant had few FK2 at 60 min (Fig. 9, a and b).
To further confirm this observation, ChIP analysis with the FK2
Figure 8. p400 is required to recruit 53BP1 and brca1 to DSBs. 293T cells
expressing p400 or p400ATPase were irradiated (2Gy). At the indicated
times, cells were fixed and processed by immunofluorescent staining to
detect either brca1 (a) or 53BP1 (b) foci. Cells with >5 foci were counted,
with an average of 100 cells per slide. Experiments represent at least three
independent replicates. Results ± SE (n = 100). (c) 293T cells expressing
p400 or p400ATPase were irradiated (2Gy). At the indicated times, cells
were fixed and processed by immunofluorescent staining to detect brca1,
53BP1 foci, and -H2AX. Bar, 5 µm.

Page 9

39
p400 regulates chromatin ubiquitination • Xu et al.
p400-dependent remodeling at sites far from the DSB, or is a
consequence of chromatin changes at the DSB that propagate
across the entire chromatin is not currently known.
Several lines of evidence suggest that p400, Trrap, and
Tip60 function together as components of the NuA4 complex to
alter nucleosome stability during DSB repair. First, although p400,
therefore likely to function primarily within the chromatin do-
mains adjacent to the DSB, where it functions to destabilize
nucleosome structures. However, our results do not exclude
the possibility that a fraction of the histones removed by NaCl
treatment are derived from regions that are remote from the
DSB, or even on adjacent chromosomes. Whether this reflects
Figure 9. p400 is required for RNF8-dependent ubiquitination of the chromatin. (a) 293T cells expressing p400 or p400ATPase were irradiated as indi-
cated. Cells were fixed and processed by immunofluorescent staining using FK2 antibody. Bar, 5 µm. (b) 293T cells expressing p400 or p400ATPase were
irradiated (2Gy). Immunofluorescent staining using the FK2 antibody was used to detect proteins ubiquitinated by RNF8. Cells with >5 foci per cell were
counted, with an average of 100 cells per slide. Results ± SE (n = 100). (c) 293T cells expressing p400 or p400ATPase were transiently transfected with
vector or p84-ZFN. 18 h later, ChIP assays using the FK2 antibody and primer pairs located at 3.5 kb, 1.5 kb, 0.5 kb, 0.5 kb, 1.5 kb, and 3.5 kb
were performed. Results ± SE (n = 3). (d) 293T cells expressing p400 or p400ATPase were irradiated (2Gy), fixed, and immunofluorescent staining to detect
RNF8 and FK2 performed. Bar, 5 µm. (e) Quantitation of FK2 and RNF8 foci in p400 and p400ATPase cells from d. Cells with >5 foci were counted, with
an average of 100 cells per slide counted. Experiments represent at least three independent replicates. Results ± SE (n = 100).

Page 10

JCB • VOLUME 191 • NUMBER 1 • 2010 40
to DSBs was abolished. This clearly demonstrates that the de-
creased nucleosome stability and associated changes in chro-
matin architecture caused by p400 are required for RNF8 to
access and ubiquitinate the chromatin-associated proteins, and
to promote recruitment of brca1 to DSBs.
Although p400 was essential to recruit brca1 to DSBs, loss
of p400 delayed (but did not abolish) the accumulation of 53BP1
at DSBs, indicating multiple pathways for recruitment of 53BP1 to
damaged chromatin. Although RNF8 plays a key role in recruit-
ing 53BP1 to DSBs (Huen et al., 2007; Kolas et al., 2007; Mailand
et al., 2007), the positioning of 53BP1 at DSBs may also involve
direct binding of 53BP1 to H4K20me2 (Sanders et al., 2004;
Botuyan et al., 2006). The p400-mediated decrease in nucleo-
some stability may therefore facilitate both ubiquitination (and
potentially sumoylation; Galanty et al., 2009; Morris et al., 2009)
of the chromatin, as well as exposing buried H4K20me2 sites that
facilitate the recruitment of 53BP1 to DSBs. Although RNF8 ubiq-
uitinates H2A and other proteins (Huen et al., 2007; Kolas et al.,
2007; Mailand et al., 2007; Doil et al., 2009; Stewart et al., 2009),
the exact target for RNF8 on the chromatin is not clear. p400-
mediated changes in nucleosome structure may therefore also func-
tion to recruit (or evict) proteins from the chromatin, including novel
proteins that are targets for either ubiquitination or sumoylation.
The key role for p400 in altering nucleosome stability at
DSBs predicts a central role for p400 in DNA repair. In fact,
p400ATPase cells exhibited both increased sensitivity to IR and in-
creased accumulation of chromosome aberrations. This is con-
sistent with previous reports indicating that the p400/Tip60
ratio plays a key role in restraining proliferation of tumor cells,
and that deregulation of this balance in colorectal cancer cells
contributes to loss of the oncogene-induced DNA damage response
(Mattera et al., 2009). p400 therefore represents a new class of
DNA damage response proteins involved in regulating chro-
matin structure at DSBs and in facilitating efficient DSB repair.
The results suggest the following model. Detection of
DSBs leads to the phosphorylation of H2AX and deposition of
mdc1 onto the chromatin. Subsequently, p400, Trrap, and Tip60
are recruited to mdc1, most likely as subunits of the NuA4 com-
plex. p400’s ATPase activity then disrupts the local histone–
DNA interactions, altering the nucleosome architecture and
facilitating the hyperacetylation of H4 by Tip60. This results in
destabilization of the nucleosomes, and promotes unpacking of
stacked nucleosome arrays. The overall result is to shift the local
chromatin structure into a relaxed, more open conformation.
Further, these regions of decreased nucleosome stability were
preferentially located in regions of high -H2AX density, sug-
gesting that -H2AX focal regions surrounding DSBs represent
relaxed, open chromatin domains. These open chromatin struc-
tures then expose RNF8 ubiquitination substrates on the chro-
matin (Huen et al., 2007; Kolas et al., 2007; Mailand et al., 2007)
as well as exposing cryptic histone methylation sites, such as
H4K20me2 (Sanders et al., 2004), which leads to recruitment of
the brca1 and 53BP1 proteins to the DSB. The overall outcome is
to facilitate DNA repair by increasing the mobility of the nucleo-
somes adjacent to the DSBs, promoting the post-translational
modification of histones, and directing the recruitment and re-
tention of protein factors at DSBs.
Tip60, and Trrap can function individually in the cell, they are
only found together as components of NuA4 (Doyon and Côté,
2004; Doyon et al., 2004). Second, yeast NuA4 is recruited to
DSBs (Downs et al., 2004). Third, the acetyltransferase activity
of the p400 complex was increased by DNA damage, indicating
a functional interaction between p400 and Tip60 during DSB
repair. Collectively, we propose that p400, Trrap, and Tip60 are
recruited to DSBs as components of the NuA4 complex. This
would then focus the catalytic activities of p400 and Tip60 onto
spatially confined regions of chromatin, allowing them to alter
the local nucleosome architecture.
Significant insight into how p400’s ATPase activity and
Tip60’s acetyltransferase activity co-coordinately regulate nucleo-
some stability was obtained. Tip60 acetylates H4 (Bird et al.,
2002; Downs et al., 2004; Doyon et al., 2004; Murr et al., 2006)
and H2AX (Kusch et al., 2004; Jha et al., 2008) after DNA dam-
age. However, the p400-dependent decrease in nucleosome sta-
bility did not require acetylation of H2AX by Tip60 (Kusch et al.,
2004; Jha et al., 2008). Instead, the decreased nucleosome stabil-
ity was associated with increased acetylation of histone H4 at
DSBs by Tip60. Histone acetylation may alter nucleosome struc-
ture through at least 2 mechanisms—charge neutralization and
the recruitment of chromatin-modifying proteins (Choi and Howe,
2009). Acetylation of the N terminus of histone H4 can induce
small changes in nucleosome structure (Tóth et al., 2006; Ferreira
et al., 2007), potentially weakening the DNA–histone interaction
(Wang and Hayes, 2008). Further, acetylation of H4 on lysine 16
(Dion et al., 2005; Shogren-Knaak et al., 2006), a site acetylated
by Tip60 (Bird et al., 2002; Doyon et al., 2004), specifically in-
hibits nucleosome packing. The ability of Tip60 to acetylate H4
at DSBs may reduce both the stability of the histone–DNA inter-
action within nucleosomes and facilitate the unpacking of higher
order nucleosome arrays (Kusch et al., 2004; Brower-Toland
et al., 2005; Murr et al., 2006; Shogren-Knaak et al., 2006). This
process also required the ATPase activity of the p400 motor pro-
tein (Doyon et al., 2004). p400 has histone exchange activity, in-
cluding the ability to exchange H2A.Z (Fuchs et al., 2001; Kusch
et al., 2004; Gévry et al., 2007), but does not function to evict
nucleosomes from the chromatin. The decrease in nucleosome
stability after DNA damage therefore reflects increased acetyla-
tion of histones, including H4, by Tip60, which promotes both
unpacking of higher order nucleosome arrays by p400 as well
as reducing the strength of histone–DNA interactions within
nucleosomes. p400 and Tip60 therefore function together to
alter chromatin structure and facilitate repair of DSBs.
The p400-mediated alterations in chromatin structure
were essential to recruit brca1 and 53BP1 to DSBs. This is con-
sistent with a previous report that Trrap is also required to locate
brca1 and 53BP1 to DSBs (Murr et al., 2006). Recruitment of
brca1 to DSBs also involves the RNF8 (and RNF168) ubiquitin
ligases, which ubiquitinate histones and other chromatin targets
at DSBs (Huen et al., 2007; Kolas et al., 2007; Mailand et al.,
2007; Doil et al., 2009). This ubiquitination creates a binding
site for the ubiquitin-interacting domain of RAP80, facilitating
the recruitment of the RAP80–abraxas–brca1 complex to DSBs.
In the absence of the p400-mediated decrease in nucleosome
stability, RNF8-dependent ubiquitination and recruitment of brca1

Page 11

41p400 regulates chromatin ubiquitination • Xu et al.
Hepes, pH 7.9. For immunoprecipitation, 4 × 106 cells were resuspended
in 1 ml NETN buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA,
1% NP-40, 1 mM DTT, and 1 mM PMSF) for 30 min, followed by centrifu-
gation at 13,000 g for 15 min. The supernatant was precleared with Pro-
tein A/G plus beads (Santa Cruz Biotechnology, Inc.) at 4°C for 30 min.
1–5 µg antibody and 30 µl Protein A/G plus beads (Santa Cruz Biotech-
nology, Inc.) were added, incubated for 3 h, washed four times in NETN
buffer containing 0.5% NP-40, and bound proteins eluted in SDS sample
buffer. For Western blots, equal amounts of protein (determined using the
DC Protein Assay kit; Bio-Rad Laboratories) were separated by SDS-PAGE
and transferred to PVDF membranes. Membranes were blocked with 5%
nonfat dry milk, incubated with primary antibodies for 1 h, and washed
in TBST (20 mM Tris, pH 7.5, 137 mM NaCl, and 0.1% Tween 20) fol-
lowed by goat anti–mouse IR Dye-800CW– or goat anti–rabbit IR Dye-
680CW–conjugated secondary antibodies (Li-Cor Inc.). Imaging was
performed using the Li-Cor Odyssey Near Infra Red System, and analyzed
using the Odyssey software system 3.0 package (Li-Cor Inc.).
For HAT assays, cells were lysed in buffer C (20 mM Hepes, pH 7.5,
150 mM NaCl, 0.2% Tween 20, 1.5 mM MgCl2, 1 mM EDTA, 2 mM DTT,
50 mM NaF, 500 µM Na3VO4, and 1 mM PMSF) and cleared lysates were
immunoprecipitated with p400 antibody (Bethyl Laboratories, Inc.). Immuno-
precipitates were washed twice in HAT assay buffer (50 mM Tris, pH 8,
10% glycerol, 0.1 mM EDTA, and 1 mM DTT) and incubated in 60 µl of
HAT assay buffer containing 100 µM acetylCoA and 0.5 µg biotinylated
histone H4 peptide for 30 min at 30°C. An aliquot of the reaction was
immobilized onto streptavidin plates and acetylation detected by ELISA as
described previously by us (Sun et al., 2005, 2007).
Nucleosome stability assay
Cells were harvested and washed twice in ice-cold PBS by centrifugation
at 1,000 rpm. Cell pellet was resuspended completely in 500 µl buffer A
(20 mM Hepes, pH 7.9, 0.5 mM DTT, 1 mM PMSF, 1.5 mM MgCl2, and
0.1% Triton) containing 1.0 M NaCl. Cells were incubated for 40 min at
4°C with constant agitation. Samples were then centrifuged at 100,000 g
(Ultracentrifuge; Beckman Coulter) for 20 min, and the supernatant, con-
taining released histones, retained for further analysis. In some experi-
ments, cells were first incubated in buffer A with 0.15 NaCl to remove cell
membrane and cytosolic contents. The nuclear fraction was then collected
by centrifugation and the nuclear pellet resuspended in buffer A plus 1.0 M
NaCl and processed as described above. Essentially identical results were
obtained using both methods.
Quantitation of H2AX and -H2AX levels
To calculate the relative levels of H2AX and -H2AX in the NaCl extracts, serial
dilutions of total histones were separated by SDS-PAGE. Western blot analysis
was then performed using H2AX and -H2AX primary antibodies followed by
goat anti–mouse IR Dye-800CW– or goat anti–rabbit IR Dye-680CW–
conjugated secondary antibodies. Infrared signals were scanned into the
Li-Cor Odyssey Near Infra Red System, and the Li-Cor software package used
to establish linear ranges for signal detection of H2AX and -H2AX. Calibra-
tion curves are shown in Fig. S1 a, and were used to determine the relative
ratio of -H2AX/H2AX in both the total cell extracts and in the NaCl extract.
ChIP assays
Chromatin immunoprecipitation (ChIP) assays used the SimpleChIP Enzy-
matic Chromatin IP kit (Cell Signaling Technology). In brief, cells were fixed
in 1% methanol-free formaldehyde for 10 min and glycine added to block
the reaction. Cells were washed twice in PBS, lyzed in ChIP buffer (Cell Sig-
naling Technology), and sonicated (Sonic 250; Thermo Fisher Scientific) to
generate DNA fragments with lengths between 200 and 1,000 bp. Insolu-
ble debris was removed by centrifugation. A portion of each supernatant
was diluted in ChIP elution buffer and digested with proteinase K at 65°C for
2 h and the released DNA fragments isolated by spin columns. The DNA
was quantitated by RT-PCR using primers (listed below) to GAPDH (Input
DNA). The remaining supernatant was diluted in ChIP buffer and equivalent
amounts of input DNA incubated with primary antibody (2 h at 4°C) fol-
lowed by collection of immune complexes on protein G–Agarose beads pre-
coated with sperm DNA. Immune complexes were washed in low and high
salt ChIP buffer (Cell Signaling Technology), the protein–DNA complex
eluted, and the DNA–protein cross-links reversed by addition of NaCl and
heating at 65°C for 2 h. After proteinase K digestion, DNA was purified
using the spin column and then quantitated by qPCR using a DNA Engine
Real-Time PCR machine (Bio-Rad Laboratories).
PCR amplification protocol: 95°C for 5 min; followed by 33 cycles
of 95°C for 30 s; 55°C for 30 s; 72°C for 30 s followed by 72°C for 5 min.
Serial dilutions of the starting material were used to determine the linear
In conclusion, we provide the first direct evidence for
altered nucleosome stability at DSBs, and have identified key
players in this process—p400, Trrap, and Tip60. Further, these
results demonstrate that changes in nucleosome stability at
DSBs can regulate both ubiquitination of chromatin by RNF8
and potentially modulate access to histone modifications, such
as histone methylation, which are buried within the packed
nucleosome arrays that constitute chromatin fibers. Finally, these
results identify p400 as a novel DNA damage response protein
that is critical in facilitating the accumulation of the brca1 protein
at sites of DNA damage.
Materials and methods
Cell culture, plasmids, and transfection
293T cells were maintained in DME supplemented with 10% fetal bovine
serum. Tip60wt and Tip60HD cells, shp400 cells, shTrrap cells, and HCT116shRad50
cells were maintained as described previously (Chan et al., 2005; Sun et al.,
2005; Zhong et al., 2005). GM5849 A-T cells complemented by expression
of ATM are described in Sun et al. (2005, 2009). H2AX/ MEFs cells were
maintained in DME supplemented with 15% fetal bovine serum, 0.8 mM
-mercaptoethanol, and 1% l-glutamine. Cells were irradiated using a Cs137
irradiator and clonogenic cell survival monitored as in Sun et al. (2005). Aver-
ages were calculated from three independent plates, and expressed plus or
minus the standard deviation (SD). To measure chromosome aberrations,
HeLaS3 cells were irradiated (2Gy) and allowed to recover for 14 h. Colce-
mid was added, and metaphase spreads prepared as described in Yang
et al. (2001). Metaphase spreads (at least 50) were scored manually under
the microscope for aberrations, with the observer blinded to the experimen-
tal conditions. Statistical analysis was performed using the Analysis ToolPak
program of Microsoft Excel to establish statistical significance.
The p400ATPase mutant was constructed using pCMV-Flag-EP400
(obtained from Chan et al., 2005) and the primer pairs: 5-GATGAAGCT-
GGGCTGGGTGCAACAGTGCAGATCATTGC-3; 5-GCAATGATCTGCA-
CTGTTGCACCCAGCCCAGCTTCATC-3, which introduces the mutation
K1085L into the ATPase domain of p400 (the p400 sequence can be found
at GenBank/EMBL/DDBJ under accession no. NP_056224). Mutagenesis
of p400 and H2AX was performed using the QuikChange Site-Directed
Mutagenesis kit (Agilent Technologies). 293T, HeLa, and MEF cells were
transfected using Lipofectamine 2000 according to the manufacturer’s in-
structions (Invitrogen), and selected using puromycin. H2AX was inserted
into plasmid pIRESpuro3 (Takara Bio Inc.), and wild-type H2AX (H2AXwt),
H2AX with an alanine mutation at lysine 5 (H2AXK5A), or H2AX with all
four N-terminal lysine residues mutated to arginine (H2AXK->R) expressed in
H2AX/ MEFs. 293T lentiviral mdc1 shRNA clones and GFP control shRNA
were obtained from The RNAi Consortium (Broad Institute, Cambridge, MA).
Plasmids were packaged with VSV-G expressing constructs phCMV-G and
PCMV8.2 plasmid and transfected into 293T cells using Lipofectamine 2000.
After 48 h, viral particles were harvested from the culture medium and used
to prepare stable cell lines expressing GFP or mdc1 shRNA after selection
with puromycin. siRNAs targeting RNF8 (#1: 5-UUCUCCUUGGGCUAUC-
UCCAAACCC-3; and #2: 5-GGAGAAUGCGGAGUAUGAAUAUGAA-3)
were transiently transfected into 293T cells using Lipofectamine 2000 (Invit-
rogen) as described previously by us (Sun et al., 2005). The p84-ZFN vector
AAVS1 was transfected into cells using Lipofectamine 2000.
Cell lysates, immunoprecipitation, and Western blot analysis
Antibodies used: p400 (Bethyl Laboratories, Inc.); H2AX, 53BP1 (Cell
Signaling Technology); -H2AX, Tip60, FK2, H2B, acetylated H4 (Millipore);
H3, H4, RNF8 (Abcam); ATM, PC116, brca1 (EMD); ATM2C1 (GeneTex);
pS1981-ATM (R&D Systems); RNF8 antibody for foci experiment was a
generous gift from Junjie Chen (MD Anderson Cancer Center, University
of Texas, Houston, TX). To extract total histones, cells were resuspended in
500 µl buffer A (20 mM Hepes, pH 7.9, 0.5 mM DTT, 1 mM PMSF, 1.5 mM
MgCl2, and 0.1% Triton) containing 0.4 M NaCl and centrifuged at 12,000 g
for 5 min. The pellet was resuspended in buffer B (10 mM Hepes, pH 7.9,
10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, and 1 mM PMSF) and 2.0N
sulfuric acid added to a final concentration of 0.4N. Samples were
cleared by centrifugation (12,000 g) and histones precipitated with 20%
trichloroacetic acid for 30 min. Histones were collected by centrifugation
(12,000 g for 15 min), washed once in acetone, and resuspended in 10 mM

Page 12

JCB • VOLUME 191 • NUMBER 1 • 2010 42
Botuyan, M.V., J. Lee, I.M. Ward, J.E. Kim, J.R. Thompson, J. Chen, and G.
Mer. 2006. Structural basis for the methylation state-specific recognition
of histone H4-K20 by 53BP1 and Crb2 in DNA repair. Cell. 127:1361–
1373. doi:10.1016/j.cell.2006.10.043
Brower-Toland, B., D.A. Wacker, R.M. Fulbright, J.T. Lis, W.L. Kraus, and M.D.
Wang. 2005. Specific contributions of histone tails and their acetylation
to the mechanical stability of nucleosomes. J. Mol. Biol. 346:135–146.
doi:10.1016/j.jmb.2004.11.056
Brunet, E., D. Simsek, M. Tomishima, R. DeKelver, V.M. Choi, P. Gregory, F.
Urnov, D.M. Weinstock, and M. Jasin. 2009. Chromosomal transloca-
tions induced at specified loci in human stem cells. Proc. Natl. Acad. Sci.
USA. 106:10620–10625. doi:10.1073/pnas.0902076106
Cairns, B.R. 2005. Chromatin remodeling complexes: strength in diversity,
precision through specialization. Curr. Opin. Genet. Dev. 15:185–190.
doi:10.1016/j.gde.2005.01.003
Carrier, F., P.T. Georgel, P. Pourquier, M. Blake, H.U. Kontny, M.J. Antinore, M.
Gariboldi, T.G. Myers, J.N. Weinstein, Y. Pommier, and A.J. Fornace Jr.
1999. Gadd45, a p53-responsive stress protein, modifies DNA accessibil-
ity on damaged chromatin. Mol. Cell. Biol. 19:1673–1685.
Chan, H.M., M. Narita, S.W. Lowe, and D.M. Livingston. 2005. The p400 E1A-
associated protein is a novel component of the p53 --> p21 senescence
pathway. Genes Dev. 19:196–201. doi:10.1101/gad.1280205
Choi, J.K., and L.J. Howe. 2009. Histone acetylation: truth of consequences?
Biochem. Cell Biol. 87:139–150. doi:10.1139/O08-112
Dion, M.F., S.J. Altschuler, L.F. Wu, and O.J. Rando. 2005. Genomic character-
ization reveals a simple histone H4 acetylation code. Proc. Natl. Acad.
Sci. USA. 102:5501–5506. doi:10.1073/pnas.0500136102
Doil, C., N. Mailand, S. Bekker-Jensen, P. Menard, D.H. Larsen, R. Pepperkok, J.
Ellenberg, S. Panier, D. Durocher, J. Bartek, et al. 2009. RNF168 binds and
amplifies ubiquitin conjugates on damaged chromosomes to allow accumula-
tion of repair proteins. Cell. 136:435–446. doi:10.1016/j.cell.2008.12.041
Downs, J.A., S. Allard, O. Jobin-Robitaille, A. Javaheri, A. Auger, N. Bouchard,
S.J. Kron, S.P. Jackson, and J. Côté. 2004. Binding of chromatin-modifying
activities to phosphorylated histone H2A at DNA damage sites. Mol.
Cell. 16:979–990. doi:10.1016/j.molcel.2004.12.003
Doyon, Y., and J. Côté. 2004. The highly conserved and multifunc-
tional NuA4 HAT complex. Curr. Opin. Genet. Dev. 14:147–154.
doi:10.1016/j.gde.2004.02.009
Doyon, Y., W. Selleck, W.S. Lane, S. Tan, and J. Côté. 2004. Structural
and functional conservation of the NuA4 histone acetyltransfer-
ase complex from yeast to humans. Mol. Cell. Biol. 24:1884–1896.
doi:10.1128/MCB.24.5.1884-1896.2004
Ferreira, H., J. Somers, R. Webster, A. Flaus, and T. Owen-Hughes. 2007. Histone
tails and the H3 alphaN helix regulate nucleosome mobility and stability.
Mol. Cell. Biol. 27:4037–4048. doi:10.1128/MCB.02229-06
Fuchs, M., J. Gerber, R. Drapkin, S. Sif, T. Ikura, V. Ogryzko, W.S.
Lane, Y. Nakatani, and D.M. Livingston. 2001. The p400 com-
plex is an essential E1A transformation target. Cell. 106:297–307.
doi:10.1016/S0092-8674(01)00450-0
Galanty, Y., R. Belotserkovskaya, J. Coates, S. Polo, K.M. Miller, and S.P.
Jackson. 2009. Mammalian SUMO E3-ligases PIAS1 and PIAS4 pro-
mote responses to DNA double-strand breaks. Nature. 462:935–939.
doi:10.1038/nature08657
Gévry, N., H.M. Chan, L. Laflamme, D.M. Livingston, and L. Gaudreau. 2007.
p21 transcription is regulated by differential localization of histone H2A.
Z. Genes Dev. 21:1869–1881. doi:10.1101/gad.1545707
Goodarzi, A.A., A.T. Noon, D. Deckbar, Y. Ziv, Y. Shiloh, M. Löbrich, and
P.A. Jeggo. 2008. ATM signaling facilitates repair of DNA double-
strand breaks associated with heterochromatin. Mol. Cell. 31:167–177.
doi:10.1016/j.molcel.2008.05.017
Gorrini, C., M. Squatrito, C. Luise, N. Syed, D. Perna, L. Wark, F. Martinato, D.
Sardella, A. Verrecchia, S. Bennett, et al. 2007. Tip60 is a haplo-insufficient
tumour suppressor required for an oncogene-induced DNA damage re-
sponse. Nature. 448:1063–1067. doi:10.1038/nature06055
Hickson, I., Y. Zhao, C.J. Richardson, S.J. Green, N.M. Martin, A.I. Orr, P.M.
Reaper, S.P. Jackson, N.J. Curtin, and G.C. Smith. 2004. Identification and
characterization of a novel and specific inhibitor of the ataxia-telangiectasia
mutated kinase ATM. Cancer Res. 64:9152–9159. doi:10.1158/0008-
5472.CAN-04-2727
Hockemeyer, D., F. Soldner, C. Beard, Q. Gao, M. Mitalipova, R.C. DeKelver,
G.E. Katibah, R. Amora, E.A. Boydston, B. Zeitler, et al. 2009. Efficient
targeting of expressed and silent genes in human ESCs and iPSCs
using zinc-finger nucleases. Nat. Biotechnol. 27:851–857. doi:10.1038/
nbt.1562
Huen, M.S., R. Grant, I. Manke, K. Minn, X. Yu, M.B. Yaffe, and J. Chen.
2007. RNF8 transduces the DNA-damage signal via histone ubiquity-
lation and checkpoint protein assembly. Cell. 131:901–914. doi:10.1016/
j.cell.2007.09.041
range of PCR amplification rate before choosing the volume of extracted
DNA used in the reaction. GAPDH was used to standardize amounts of
input DNA for each reaction. Standard controls included immunoprecipita-
tion with IgG and qPCR of immunoprecipitated DNA with GAPDH, both of
which yielded essentially no signal. Relative folds of enrichment were opti-
mized by the input control and expressed as IP/Input DNA. The relative
increase in signal after cutting by the p84-ZFN was calculated as [IPZFN/
InputZFN]/[IPControl/InputControl]. All ChIP assays were repeated at least twice,
and individual qPCR reactions were performed in triplicate, with results
presented ±SD. PCR primers: 0.5 kb (5-TGGGTTCCCTTTTCCTTCTC-3
and 5-GTCCAGGCAAAGAAAGCAAG-3); +0.5 kb (5-ATGGTGCG-
TCCTAGGTGTTC-3 and 5-CCAAGGACTCAAACCCAGAA-3); 1.5 kb
(5-GGGGCAGTCTGCTATTCATC-3 and 5-CGATGCACACTGGGA-
AGTC-3); +1.5 kb (5-CCTCAGCTCCAGTTCAGGTC-3 and 5-GGCTGT-
CACACTCCAGTTCA-3); 3.5 kb (5-CCGAGATCACATCACTGCAC-3
and 5-GGAAAGAGGAGGGAAGAGGA-3); +3.5 kb (5-TCGCCAGT-
GCTTTTTCTTTT-3 and 5-GTTGGGGGATGATGAAAATG-3); GAPDH
primers: (5-CCTGACCTGCCGTCTAGAAAA-3 and 5-CTCCGACGCCT-
GCTTCAC-3); DSB primers: (5-CGGTTAATGTGGCTCTGGTT-3 and
5-ACAGGAGGTGGGGGTTAGAC-3); Chr 6 primers: (5-CATGGTCGT-
CAACCAAATGA-3 and 5-CCCAGAATCTTCTGACCTGCT-3).
Immunofluorescence
Cells (on cover slides) were fixed in phosphate-buffered saline (PBS) contain-
ing 4% paraformaldehyde, blocked with 2% fetal bovine serum for 10 min,
and permeabilized in 0.2% bovine serum albumin containing 0.2% saponin
for 15 min. After a 1-h incubation with primary antibody, cells were washed
three times with 0.2% Tween 20 and incubated for 1 h in secondary anti-
body (conjugated to either Texas red or FITC; Santa Cruz Biotechnology,
Inc.). Slides were mounted with Fluoromount-G (SouthernBiotech) and im-
aged at room temperature. Images were collected with a microscope (Axio-
Imager Z1; Carl Zeiss, Inc.) equipped with a color digital camera (Axiocam
MRc Rev.3; Carl Zeiss, Inc.) and a Plan Apochromat oil M27 lens (63x, NA
1.4). Acquisition software and image processing used the AxioVision soft-
ware package (Carl Zeiss, Inc.).
RT-PCR for mdc1 mRNA levels
Cells were lyzed in 1 ml Triazol and 200 µl chloroform, centrifuged, and the
upper layer precipitated with isopropanol. Washed pellets were digested
with DNase1, precipitated (3 M sodium acetate), and resuspended in water.
1 µg RNA and 50 ng random primers were heated to 70°C, chilled, and
1 µM dNTPs, 1 mM DTT, and 100 units of MMLV reverse transcription added
to generate cDNA. RT-qPCR was performed using primers 5-CTGGGTAG-
GTTCATCCTCCA-3 and 5-GGAAAGGGTGTCATTCTGGA-3 and GAPDH
primers as an internal control.
Online supplemental material
Fig. S1 demonstrates that the MRN complex and the ATM kinase are not
required for reduced nucleosome stability. Fig. S2 demonstrates efficient si-
lencing of p400 and Trrap expression, as well as expression of p400ATPase
and Tip60HD in cells. Fig. S3 demonstrates that p84-ZFN generates DSBs
in a range of cell lines, and activates the DNA damage response, includ-
ing -H2AX production. Fig. S4 demonstrates that p400-mediated nucleo-
some stability requires both H2AX and mdc1, but does not require the
ubiquitin ligase activity of RNF8. Online supplemental material is available
at http://www.jcb.org/cgi/content/full/jcb.201001160/DC1.
We thank Junjie Chen for RNF8 constructs and antibodies, Ralph Scully for
mdc1/ MEFs, Homan Chan for p400 shRNA and Trrap shRNA cells, and
Fyodor Urnov for the AAVS1.
This work was supported by grants from the National Cancer Institute
(CA64585 and CA93602), the DOD Breast Cancer Program, and by a Na-
tional Cancer Institute training grant to Y. Sun (T32 CA09078).
Submitted: 28 January 2010
Accepted: 1 September 2010
References
Bird, A.W., D.Y. Yu, M.G. Pray-Grant, Q. Qiu, K.E. Harmon, P.C. Megee, P.A.
Grant, M.M. Smith, and M.F. Christman. 2002. Acetylation of histone
H4 by Esa1 is required for DNA double-strand break repair. Nature.
419:411–415. doi:10.1038/nature01035
Bonner, W.M., C.E. Redon, J.S. Dickey, A.J. Nakamura, O.A. Sedelnikova,
S. Solier, and Y. Pommier. 2008. GammaH2AX and cancer. Nat. Rev.
Cancer. 8:957–967. doi:10.1038/nrc2523

Page 13

43p400 regulates chromatin ubiquitination • Xu et al.
regions of human chromatin. Biochemistry. 17:2377–2386. doi:10.1021/
bi00605a020
Spycher, C., E.S. Miller, K. Townsend, L. Pavic, N.A. Morrice, P. Janscak, G.S.
Stewart, and M. Stucki. 2008. Constitutive phosphorylation of MDC1
physically links the MRE11-RAD50-NBS1 complex to damaged chro-
matin. J. Cell Biol. 181:227–240. doi:10.1083/jcb.200709008
Stewart, G.S., S. Panier, K. Townsend, A.K. Al-Hakim, N.K. Kolas, E.S. Miller,
S. Nakada, J. Ylanko, S. Olivarius, M. Mendez, et al. 2009. The RIDDLE
syndrome protein mediates a ubiquitin-dependent signaling cascade at sites
of DNA damage. Cell. 136:420–434. doi:10.1016/j.cell.2008.12.042
Stiff, T., M. O’Driscoll, N. Rief, K. Iwabuchi, M. Löbrich, and P.A. Jeggo.
2004. ATM and DNA-PK function redundantly to phosphorylate
H2AX after exposure to ionizing radiation. Cancer Res. 64:2390–2396.
doi:10.1158/0008-5472.CAN-03-3207
Stucki, M., J.A. Clapperton, D. Mohammad, M.B. Yaffe, S.J. Smerdon, and S.P.
Jackson. 2005. MDC1 directly binds phosphorylated histone H2AX to
regulate cellular responses to DNA double-strand breaks. Cell. 123:1213–
1226. doi:10.1016/j.cell.2005.09.038
Sun, Y., X. Jiang, S. Chen, N. Fernandes, and B.D. Price. 2005. A role for the Tip60
histone acetyltransferase in the acetylation and activation of ATM. Proc.
Natl. Acad. Sci. USA. 102:13182–13187. doi:10.1073/pnas.0504211102
Sun, Y., Y. Xu, K. Roy, and B.D. Price. 2007. DNA damage-induced acetylation
of lysine 3016 of ATM activates ATM kinase activity. Mol. Cell. Biol.
27:8502–8509. doi:10.1128/MCB.01382-07
Sun, Y., X. Jiang, Y. Xu, M.K. Ayrapetov, L.A. Moreau, J.R. Whetstine, and B.D.
Price. 2009. Histone H3 methylation links DNA damage detection to
activation of the tumour suppressor Tip60. Nat. Cell Biol. 11:1376–1382.
doi:10.1038/ncb1982
Tóth, K., N. Brun, and J. Langowski. 2006. Chromatin compaction at the mono-
nucleosome level. Biochemistry. 45:1591–1598. doi:10.1021/bi052110u
Tsukuda, T., A.B. Fleming, J.A. Nickoloff, and M.A. Osley. 2005. Chromatin
remodelling at a DNA double-strand break site in Saccharomyces cerevi-
siae. Nature. 438:379–383. doi:10.1038/nature04148
Urnov, F.D., J.C. Miller, Y.L. Lee, C.M. Beausejour, J.M. Rock, S. Augustus,
A.C. Jamieson, M.H. Porteus, P.D. Gregory, and M.C. Holmes. 2005.
Highly efficient endogenous human gene correction using designed zinc-
finger nucleases. Nature. 435:646–651. doi:10.1038/nature03556
van Attikum, H., O. Fritsch, B. Hohn, and S.M. Gasser. 2004. Recruitment of the
INO80 complex by H2A phosphorylation links ATP-dependent chroma-
tin remodeling with DNA double-strand break repair. Cell. 119:777–788.
doi:10.1016/j.cell.2004.11.033
von Holt, C., W.F. Brandt, H.J. Greyling, G.G. Lindsey, J.D. Retief, J.D.
Rodrigues, S. Schwager, and B.T. Sewell. 1989. Isolation and charac-
terization of histones. Methods Enzymol. 170:431–523. doi:10.1016/
0076-6879(89)70061-6
Wang, X., and J.J. Hayes. 2008. Acetylation mimics within individual core
histone tail domains indicate distinct roles in regulating the stabil-
ity of higher-order chromatin structure. Mol. Cell. Biol. 28:227–236.
doi:10.1128/MCB.01245-07
Yang, Y., Y. Kuang, R. Montes De Oca, T. Hays, L. Moreau, N. Lu, B. Seed,
and A.D. D’Andrea. 2001. Targeted disruption of the murine Fanconi
anemia gene, Fancg/Xrcc9. Blood. 98:3435–3440. doi:10.1182/blood
.V98.12.3435
Zhong, H., A. Bryson, M. Eckersdorff, and D.O. Ferguson. 2005. Rad50 deple-
tion impacts upon ATR-dependent DNA damage responses. Hum. Mol.
Genet. 14:2685–2693. doi:10.1093/hmg/ddi302
Ziv, Y., D. Bielopolski, Y. Galanty, C. Lukas, Y. Taya, D.C. Schultz, J. Lukas,
S. Bekker-Jensen, J. Bartek, and Y. Shiloh. 2006. Chromatin relaxation
in response to DNA double-strand breaks is modulated by a novel
ATM- and KAP-1 dependent pathway. Nat. Cell Biol. 8:870–876.
doi:10.1038/ncb1446
Ikura, T., S. Tashiro, A. Kakino, H. Shima, N. Jacob, R. Amunugama, K. Yoder,
S. Izumi, I. Kuraoka, K. Tanaka, et al. 2007. DNA damage-dependent
acetylation and ubiquitination of H2AX enhances chromatin dynamics.
Mol. Cell. Biol. 27:7028–7040. doi:10.1128/MCB.00579-07
Jha, S., E. Shibata, and A. Dutta. 2008. Human Rvb1/Tip49 is required for the
histone acetyltransferase activity of Tip60/NuA4 and for the downregula-
tion of phosphorylation on H2AX after DNA damage. Mol. Cell. Biol.
28:2690–2700. doi:10.1128/MCB.01983-07
Kimura, H., and P.R. Cook. 2001. Kinetics of core histones in living human cells:
little exchange of H3 and H4 and some rapid exchange of H2B. J. Cell
Biol. 153:1341–1353. doi:10.1083/jcb.153.7.1341
Kolas, N.K., J.R. Chapman, S. Nakada, J. Ylanko, R. Chahwan, F.D. Sweeney,
S. Panier, M. Mendez, J. Wildenhain, T.M. Thomson, et al. 2007.
Orchestration of the DNA-damage response by the RNF8 ubiquitin li-
gase. Science. 318:1637–1640. doi:10.1126/science.1150034
Kruhlak, M.J., A. Celeste, G. Dellaire, O. Fernandez-Capetillo, W.G. Müller,
J.G. McNally, D.P. Bazett-Jones, and A. Nussenzweig. 2006. Changes in
chromatin structure and mobility in living cells at sites of DNA double-
strand breaks. J. Cell Biol. 172:823–834. doi:10.1083/jcb.200510015
Kusch, T., L. Florens, W.H. Macdonald, S.K. Swanson, R.L. Glaser, J.R. Yates
III, S.M. Abmayr, M.P. Washburn, and J.L. Workman. 2004. Acetylation
by Tip60 is required for selective histone variant exchange at DNA
lesions. Science. 306:2084–2087. doi:10.1126/science.1103455
Lee, J.H., and T.T. Paull. 2005. ATM activation by DNA double-strand breaks through
the Mre11-Rad50-Nbs1 complex. Science. 308:551–554. doi:10.1126/
science.1108297
Mailand, N., S. Bekker-Jensen, H. Faustrup, F. Melander, J. Bartek, C. Lukas,
and J. Lukas. 2007. RNF8 ubiquitylates histones at DNA double-strand
breaks and promotes assembly of repair proteins. Cell. 131:887–900.
doi:10.1016/j.cell.2007.09.040
Mattera, L., F. Escaffit, M.J. Pillaire, J. Selves, S. Tyteca, J.S. Hoffmann, P.A.
Gourraud, M. Chevillard-Briet, C. Cazaux, and D. Trouche. 2009.
The p400/Tip60 ratio is critical for colorectal cancer cell proliferation
through DNA damage response pathways. Oncogene. 28:1506–1517.
doi:10.1038/onc.2008.499
Melander, F., S. Bekker-Jensen, J. Falck, J. Bartek, N. Mailand, and J. Lukas.
2008. Phosphorylation of SDT repeats in the MDC1 N terminus triggers
retention of NBS1 at the DNA damage-modified chromatin. J. Cell Biol.
181:213–226. doi:10.1083/jcb.200708210
Morris, J.R., C. Boutell, M. Keppler, R. Densham, D. Weekes, A. Alamshah, L.
Butler, Y. Galanty, L. Pangon, T. Kiuchi, et al. 2009. The SUMO modi-
fication pathway is involved in the BRCA1 response to genotoxic stress.
Nature. 462:886–890. doi:10.1038/nature08593
Murr, R., J.I. Loizou, Y.G. Yang, C. Cuenin, H. Li, Z.Q. Wang, and Z. Herceg.
2006. Histone acetylation by Trrap-Tip60 modulates loading of repair
proteins and repair of DNA double-strand breaks. Nat. Cell Biol. 8:91–
99. doi:10.1038/ncb1343
Papamichos-Chronakis, M., J.E. Krebs, and C.L. Peterson. 2006. Interplay be-
tween Ino80 and Swr1 chromatin remodeling enzymes regulates cell
cycle checkpoint adaptation in response to DNA damage. Genes Dev.
20:2437–2449. doi:10.1101/gad.1440206
Robert, F., S. Hardy, Z. Nagy, C. Baldeyron, R. Murr, U. Déry, J.Y. Masson, D.
Papadopoulo, Z. Herceg, and L. Tora. 2006. The transcriptional histone
acetyltransferase cofactor TRRAP associates with the MRN repair com-
plex and plays a role in DNA double-strand break repair. Mol. Cell. Biol.
26:402–412. doi:10.1128/MCB.26.2.402-412.2006
Rubbi, C.P., and J. Milner. 2003. p53 is a chromatin accessibility factor for
nucleotide excision repair of DNA damage. EMBO J. 22:975–986.
doi:10.1093/emboj/cdg082
Samuelson, A.V., M. Narita, H.M. Chan, J. Jin, E. de Stanchina, M.E. McCurrach,
M. Narita, M. Fuchs, D.M. Livingston, and S.W. Lowe. 2005. p400 is re-
quired for E1A to promote apoptosis. J. Biol. Chem. 280:21915–21923.
doi:10.1074/jbc.M414564200
Sanders, S.L., M. Portoso, J. Mata, J. Bähler, R.C. Allshire, and T. Kouzarides. 2004.
Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites
of DNA damage. Cell. 119:603–614. doi:10.1016/j.cell.2004.11.009
Shechter, D., H.L. Dormann, C.D. Allis, and S.B. Hake. 2007. Extraction,
purification and analysis of histones. Nat. Protoc. 2:1445–1457.
doi:10.1038/nprot.2007.202
Shogren-Knaak, M., H. Ishii, J.M. Sun, M.J. Pazin, J.R. Davie, and C.L. Peterson.
2006. Histone H4-K16 acetylation controls chromatin structure and pro-
tein interactions. Science. 311:844–847. doi:10.1126/science.1124000
Shroff, R., A. Arbel-Eden, D. Pilch, G. Ira, W.M. Bonner, J.H. Petrini, J.E.
Haber, and M. Lichten. 2004. Distribution and dynamics of chromatin
modification induced by a defined DNA double-strand break. Curr. Biol.
14:1703–1711. doi:10.1016/j.cub.2004.09.047
Smerdon, M.J., T.D. Tlsty, and M.W. Lieberman. 1978. Distribution of ultra-
violet-induced DNA repair synthesis in nuclease sensitive and resistant