DNA double-strand breaks promote methylation of

histone H3 on lysine 9 and transient formation of

repressive chromatin
Marina K. Ayrapetova,1, Ozge Gursoy-Yuzugullua,1, Chang Xua,b, Ye Xua, and Brendan D. Pricea,2
a
Department of Radiation Oncology, DanaFarber Cancer Institute, Harvard Medical School, Boston, MA 02215; and bInstitute of Radiation Medicine, Tianjin
Key Laboratory of Molecular Nuclear Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin 300192, Peoples Republic
of China

Edited by Steven Henikoff, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved May 14, 2014 (received for review February 26, 2014)

histone methylation

acetylation and ubiquitination of the chromatin and loading of

DNA repair proteins is therefore critical for DSB repair.
Activation of Tip60s acetyltransferase activity requires interaction between Tip60s chromodomain and histone H3 methylated on lysine 9 (H3K9me3) on nucleosomes at the break (4, 6).
This interaction, in combination with tyrosine phosphorylation of
Tip60 (17), increases Tip60s acetyltransferase activity and promotes acetylation of both the ATM kinase and histone H4 (46,
17). Consequently, loss of H3K9me2/3 leads to failure to activate
the ATM signaling pathway, loss of H4 acetylation during DSB
repair, disruption of heterochromatin, genomic instability, and
defective DSB repair (4, 1719). H3K9me3s therefore play a critical role in linking chromatin structure at DSBs to the activation of
DSB signaling proteins such as Tip60 and ATM.
How Tip60 gains access to H3K9me3 and how H3K9me3 levels
at DSBs are regulated is not known. H3K9me3 is concentrated
in heterochromatin domains, where it recruits HP1, kap-1, and
H3K9 methyltransferases (20, 21) to maintain the silent, compact
conformation of heterochromatin (20). This implies that Tip60s
acetyltransferase activity can only be activated at DSBs in regions
of high H3K9me3 density, such as heterochromatin. Alternatively, H3K9 methylation may be actively increased at DSBs in
regions of low H3K9me3 density to allow for Tip60 activation and
Significance
Double-strand break (DSB) repair initiates dynamic changes in
histone modifications that are required to maintain genome
stability. Methylation of histone H3 lysine-9 (H3K9me3) is
critical for activating ataxia telangiectasia-mutated (ATM) kinase, but how H3K9 methylation is regulated at DSBs is unknown. We show that a complex containing the suv39h1
methyltransferase is rapidly recruited to DSBs, where it directs
H3K9 methylation on large chromatin domains adjacent to the
DSB. This process results in transient formation of repressive
chromatin and serves to both stabilize the chromatin structure
and promote activation of DSB-signaling proteins, including ATM
kinase. Dynamic changes in H3K9 modification in euchromatin by suv39h1 are therefore one of the earliest signaling
events required for processing and remodeling of the damaged
chromatin template.

| homologous recombination

NA double-strand breaks (DSBs) are toxic and must be

repaired to maintain genomic stability. Detection of DSBs
requires recruitment of the mre11rad50nbs1 (MRN) complex
to the DNA ends (1). MRN then recruits and activates the ataxia
telangiectasia-mutated (ATM) kinase (2, 3) through a mechanism that also requires the Tip60 acetyltransferase (3). Tip60
directly acetylates and activates ATMs kinase activity (46) and
functions, in combination with MRN, to promote ATM-dependent
phosphorylation of DSB repair proteins (3), including histone
H2AX. This process creates domains of phosphorylated H2AX
(H2AX) extending for hundreds of kilobases along the chromatin (7, 8). Mdc1 then binds to H2AX, providing a landing
pad for other DSB repair proteins, including the RNF8/RNF168
ubiquitin ligases (1, 3, 9, 10). Tip60 also plays a critical role in
regulating chromatin structure at DSBs as part of the NuA4
Tip60 complex (11). NuA4-Tip60 catalyzes histone exchange (via
the p400 ATPase subunit) and acetylation of histone H4 (by Tip60)
at DSBs (1215), leading to the formation of open, flexible chromatin domains adjacent to the break (12, 13). These open chromatin structures then facilitate histone ubiquitination, the loading
of brca1 and 53BP1, and repair of the DSB (13, 16). The ordered
www.pnas.org/cgi/doi/10.1073/pnas.1403565111

Author contributions: O.G.-Y. and B.D.P. designed research; B.D.P. conceived and planned
the study, with input from M.K.A. and O.G.-Y.; M.K.A., O.G.-Y., C.X., and Y.X. performed
research; M.K.A. performed laser microirradiation, plasmid construction, and cell-based
analysis; O.G.-Y. performed the ChIP experiments with contributions from C.X.; Y.X. contributed to assay development; M.K.A., O.G.-Y., and B.D.P. analyzed data; and M.K.A.,
O.G.-Y., and B.D.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access option.
1

M.K.A. and O.G.-Y. contributed equally to this work.

To whom correspondence should be addressed. E-mail: brendan_price@dfci.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.

1073/pnas.1403565111/-/DCSupplemental.

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Dynamic changes in histone modification are critical for regulating

DNA double-strand break (DSB) repair. Activation of the Tip60
acetyltransferase by DSBs requires interaction of Tip60 with histone H3 methylated on lysine 9 (H3K9me3). However, how H3K9
methylation is regulated during DSB repair is not known. Here, we
demonstrate that a complex containing kap-1, HP1, and the H3K9
methyltransferase suv39h1 is rapidly loaded onto the chromatin at
DSBs. Suv39h1 methylates H3K9, facilitating loading of additional
kap-1/HP1/suv39h1 through binding of HP1s chromodomain to
the nascent H3K9me3. This process initiates cycles of kap-1/HP1/
suv39h1 loading and H3K9 methylation that facilitate spreading of
H3K9me3 and kap-1/HP1/suv39h1 complexes for tens of kilobases
away from the DSB. These domains of H3K9me3 function to activate the Tip60 acetyltransferase, allowing Tip60 to acetylate both
ataxia telangiectasia-mutated (ATM) kinase and histone H4. Consequently, cells lacking suv39h1 display defective activation of Tip60
and ATM, decreased DSB repair, and increased radiosensitivity. Importantly, activated ATM rapidly phosphorylates kap-1, leading to
release of the repressive kap-1/HP1/suv39h1 complex from the
chromatin. ATM activation therefore functions as a negative feedback loop to remove repressive suv39h1 complexes at DSBs, which
may limit DSB repair. Recruitment of kap-1/HP1/suv39h1 to DSBs
therefore provides a mechanism for transiently increasing the levels
of H3K9me3 in open chromatin domains that lack H3K9me3 and
thereby promoting efficient activation of Tip60 and ATM in these
regions. Further, transient formation of repressive chromatin may
be critical for stabilizing the damaged chromatin and for remodeling the chromatin to create an efficient template for the DNA
repair machinery.

efficient DSB repair in euchromatin. Understanding the dynamics of H3K9 methylation at DSBs is therefore critical to understanding how Tip60 activity is regulated by the local chromatin
architecture. Here, we show that the suv39h1 methyltransferase is
recruited to DSBs in euchromatin as part of a larger kap-1/HP1/
suv39h1 complex. Suv39h1 increases H3K9me3 at DSBs, activating Tip60s acetyltransferase activity and promoting the subsequent acetylation and activation of ATM. Further, loss of
inducible H3K9me3 at DSBs leads to defective repair and increased
radiosensitivity. Finally, loading of the kap-1/HP1/suv39h1 complex is transient, and the complex is rapidly released from the
chromatin through a negative feedback loop driven by ATMdependent phosphorylation of the kap-1 protein.
Results
Initially, we determined whether H3K9me3 participates in DSB
repair in chromatin domains that lack endogenous H3K9me2/3.
The p84zinc finger nuclease (p84-ZFN) creates a DSB in intron 1
of the PPP1R12C gene (12, 13). PPP1R12C lacks significant
H3K9me2/3 but is rich in marks associated with transcription
(ENCODE database, http://encodeproject.org/ENCODE/). Chromatin IP (ChIP) demonstrated increased phosphorylation of
H2AX (H2AX) at the p84-ZFN DSB (Fig. 1A). ChIP with
H3K9me3 antibody (Fig. S1A) demonstrated increased H3K9me3
on either side of the DSB (1.5 kb), with lower levels of H3K9me3
extending >200 kb away from the DSB (Fig. 1A). A small increase in H3K9me2 was also detected (Fig. 1B). Histone H3
levels were unchanged (Fig. 1B), indicating increased methylation
rather than changes in H3 content. Further, no change in either
H3K36me3 or H4K20me2 was seen at the DSB (Fig. S1B). Finally, H3K9me3 was not increased at a distal chromosome site
(Fig. S1C), indicating that the increased H3K9me3 is restricted to the chromatin domain adjacent to the DSB.
The H3K9 methyltransferase suv39h1 has been implicated in
DSB repair (4, 18, 19). Depletion of suv39h1 with siRNA (Fig.
S1D) significantly reduced H3K9me3 at the p84-ZFN DSB (Fig.
1C). Furthermore, ChIP demonstrated that suv39h1 was loaded
onto the chromatin at the DSB (Fig. 1D and Fig. S1E). Finally,
suv39h1 was rapidly (within 5 min) recruited to regions of DNA
damage created with laser microirradiation (Fig. 1E). The
suv39h1 methyltransferase is therefore recruited to DSBs and
increases local H3K9me3 in response to DSBs.
Interaction between Tip60 and H3K9me3 (4) promotes acetylation of ATM (5, 6, 17) and histone H4 (1214) by Tip60.
Depletion of suv39h1 reduced inducible H3K9me3 (Fig. 1C) and
inhibited acetylation of histone H4 by Tip60 (Fig. 2A). Furthermore, depletion of suv39h1 attenuated ATM activation
(Fig. S2A) and reduced phosphorylation of kap-1 (Fig. S2B).
This finding is consistent with methylation of H3K9 by suv39h1
playing an essential role in activation of Tip60s acetyltransferase
activity and the subsequent acetylation and activation of the
ATM kinase. Furthermore, cells lacking suv39h1 have increased
radiation sensitivity (Fig. 2B) and reduced homologous recombination (HR)-mediated repair (Fig. 2C). Finally, recruitment of
brca1 and RPA32 to DSBs (Fig. 2D) were reduced following loss
of suv39h1, consistent with the decrease in HR-mediated repair
in suv39h1-deficient cells (Fig. 2C). However, nonhomologous
end-joining (NHEJ) activity was not significantly altered by loss
of suv39h1 (Fig. S2C), implying that regulation of the Ku70/80
complex and DNAPKcs activity does not require H3K9 methylation. These results are consistent with previous studies demonstrating
increased genomic instability in mice and other experimental systems
(4, 18, 19) in which suv39h1 was inactivated. Methylation of
H3K9 by suv39h1 at DSBs therefore plays a critical role in activating Tip60, controlling ATM signaling, and in directing DSB
repair and maintaining genomic stability.
Two heterochromatin-binding proteins, kap-1 and HP1, are
corecruited to sites of DNA damage (22, 23), although how they
impact DSB repair is not clear. However, suv39h1 can interact with
kap-1/HP1 repressor complexes (20, 21, 24), suggesting that kap-1
and HP1 may recruit suv39h1 to DSBs. Initially, we confirmed that
9170 | www.pnas.org/cgi/doi/10.1073/pnas.1403565111

Fig. 1. Suv39h1 promotes H3K9 methylation at DSBs. (A) 293T cells transfected with p84-ZFN were processed for ChIP by using H2AX or
H3K9me3 antibodies, followed by quantitative RT-PCR (RT-qPCR) with
primer pairs located at the indicated positions. Results are fold enrichment
relative to uncut DNA, which is assigned a value of 1 (solid black line; Uncut).
Results are SD (n = 3). (B) 293T cells transfected with p84-ZFN (ZFN) or
vector (Vec) were processed for ChIP by using antibodies to H3, H2AX,
H3K9me3, or H3K9me2 and primer pairs located 1.5 kb to the right of the
DSB. Results are expressed as fold enrichment relative to uncut DNA. Results
are SD (n = 3). (C) 293T cells were transfected with nonspecific () or siRNA
targeting suv39h1 (+). Forty-eight hours later, cells were transfected with
vector (Vec) or p84-ZFN (ZFN) and processed for ChIP by using antibody
against H3K9me3 and primers located 1.5 kb to either side of the DSB.
Results are SD (n = 3). (D) 293T cells were transfected with vector (Vec) or
p84-ZFN (ZFN) and processed for ChIP with suv39h1 antibody and primers
located 1.5 kb to either side of the DSB. Results are SD (n = 3). (E ) U2OS
cells were transfected with nonspecific (control) or suv39h1-specific
(siSuv39h1) siRNA. Forty-eight hours later, focused regions of DNA damage
were produced by using a scanning laser system. Cells were fixed for immunofluorescent staining by using antibodies to H2AX (green) or suv39h1
(red). Nuclei were stained with DAPI (blue).

kap-1 interacts with the HP1, HP1, and suv39h1 and that this
interaction was not altered by DNA damage (Fig. S3 B and C).
When laser striping was used to create focused regions of DNA
damage, suv39h1 (Fig. 3A) and kap-1 (Fig. 3B) were recruited to
sites of DNA damage with similar kinetics, such that 90% of
H2AX stripes were colocalized with suv39h1 and kap-1 (Fig.
3C). Furthermore, ChIP demonstrated that HP1 was also
recruited to sites of DNA damage (Fig. 3D). Importantly,
Ayrapetov et al.

suv39h1, kap-1, and HP1 were only transiently retained at damage

sites (Fig. 3 A and B), indicating that they function during the
initial few minutes following DSB production. Furthermore, depletion of kap-1 (Fig. S3A) blocked recruitment of both suv39h1
(Fig. 3 A and C) and HP1 (Fig. 3D) to DSBs. Similarly, depletion of suv39h1 blocked recruitment of kap-1 (Fig. 3 B and C
and Fig. S3D) and HP1 (Fig. 3D) to DSBs. Loss of any one of
suv39h1, kap-1, or HP1 therefore prevents loading of all three
proteins at the DSB. Finally, depletion of kap-1, which blocks
recruitment of both suv39h1 and HP1 to DSBs, abolished the
increase in H3K9me3 at DSBs (Fig. 3E). Combining these data
with previous work demonstrating direct interaction between
kap-1, HP1 family members, and suv39h1 (2428) implies that
kap-1, HP1, and suv39h1 are recruited to DSBs as a single kap-1/
HP1/suv39h1 complex and that it is this complex that directs
H3K9 methylation at DSBs.
Next, we examined suv39h1s methyltransferase activity.
Suv39h1MD, containing a point mutation in the catalytic domain,
was efficiently incorporated into the kap-1/HP1/suv39h1 complex
(Fig. S4A). However, suv39h1MD was not recruited to regions of
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Fig. 2. Suv39h1 regulates Tip60 activity, genomic stability, and homologous

recombination (HR)-mediated repair. (A) 293T cells expressing nonspecific
shRNA (control) or shRNA targeting suv39h1 were transfected with vector
(Vec) or p84-ZFN (ZFN), followed by ChIP with antibody to H4Ac. RT-qPCR
was carried out using primer pairs located to 1.5 kb to the right of the DSB.
Results are SD (n = 3). (B) 293T cells expressing nonspecific shRNA () or
shRNA targeting suv39h1 () were irradiated, and clonogenic cell survival
assays were carried out. Results are SD (n = 3). (C) U2OS cells containing
a GFPHR reporter were stably transfected with nonspecific (NS) or shRNA
targeting suv39h1, followed by transient transfection with vector (Vec) or
the I-Sce1 endonuclease. GFP-positive cells were counted by FACS. Results
are SD (n = 4 biological replicates). (D) U2OS cells expressing nonspecific ()
or shRNA targeting suv39h1 (+) were irradiated (2 Gy), allowed to recover
for 30 min, and stained with antibodies to H2AX, brca1, or RPA32. Cells
with more than five foci were scored as positive. Results are SD (n = 3). P
value was determined by using a t test.

laser damage, and, furthermore, it blocked recruitment of kap-1

(Fig. 4A), such that <5% of H2AX stripes in suv39h1MD cells
contained either kap-1 or suv39h1 (see Fig. S4E for quantitation). Dimethyloxalylglycine (DMOG), a pan-specific inhibitor
of H3K9 demethylases (29), was then used to increase global
H3K9me3 levels (Fig. S4 B and D). DMOG rescued recruitment
of both suv39h1MD and kap-1 to regions of laser damage (Fig.
4A), such that >80% of H2AX stripes in suv39h1MD cells then
colocalized with suv39h1 and kap-1 (Fig. S4E). Further, DMOG
rescued recruitment of kap-1 and HP1, even in cells lacking
expression of suv39h1 (Fig. 4B). This finding demonstrates that
H3K9me3, rather than suv39h1, mediates retention of kap-1,
HP1, and suv39h1 on the chromatin. Because the chromodomain
of HP1 binds to H3K9me3, we deleted HP1s chromodomain
(Fig. S4D). Loss of HP1s chromodomain did not alter interaction of HP1 with either suv39h1 or kap-1 (Fig. S4 F and G).
Importantly, HP1CD was not recruited to sites of laser microirradiation and inhibited corecruitment of suv39h1 (Fig. 4C).
Furthermore, increasing H3K9me3 with DMOG did not restore
loading of HP1 or suv39h1 in HP1CD cells (Fig. 4C), demonstrating that both HP1s chromodomain and H3K9me3 are required to retain kap-1/HP1/suv39h1 at DSBs. This finding suggests
a model in which the initial positioning of kap-1/HP1/suv39h1 at
DSBs promotes H3K9 methylation by suv39h1. This priming
event then recruits additional kap-1/HP1/suv39h1 complexes,
which are retained through interaction between HP1s chromodomain and newly synthesized H3K9me3. This kap-1/HP1/suv39h1
then bridges to more distal nucleosomes, promoting additional
cycles of H3K9 methylation and kap-1/HP1/suv39h1 binding,
which spreads H3K9me3 along the chromatin away from the DSB.
Spreading therefore requires both suv39h1 (to create H3K9me3)
and HP1 (which binds to H3K9me3). Consequently, loss of HP1,
suv39h1, or kap-1 prevents spreading of H3K9me3 and therefore
loading of the complex onto the chromatin.
Kap-1, HP1, and suv39h1 are only retained at the DSB during
the first few minutes after damage (Figs. 3 and 4). Because the
ATM-dependent phosphorylation of kap-1 weakens kap-1s interaction with chromatin (30, 31), we examined whether ATM
was required to release kap-1/HP1/suv39h1 from DSBs. The
ATM inhibitor Ku-55933 (ATMi) did not alter interaction between
kap-1 and suv39h1 (Fig. S5A). Suv39h1 (Fig. 5A) and kap-1 (Fig.
5B) were recruited to DSBs in the presence of ATMi. However,
ATMi blocked release of suv39h1 and kap-1 (Fig. 5 A and B) and
increased retention of kap-1 at p84-ZFNgenerated DSBs (Fig.
S5B). Similarly, inactivation of the MRN complex, which is required for ATM activation (2), blocked phosphorylation of kap-1
by ATM (Fig. S5D) and prevented release of suv39h1 from sites
of DNA damage (Fig. S5C). Phosphorylation of serine 824 of
kap-1 by ATM weakens kap-1 interaction with chromatin (3032).
Kap-1wt, kap-1S824A, and kap-1S824D (containing a phospho-mimic)
were expressed in U2OS cells (Fig. S5E). Kap-1wt was transiently
recruited to and released from regions of DNA damage, whereas
the nonphosphorylatable kap-1S824A was recruited to DSBs but
retained at DSBs for an extended time (Fig. 5C and Fig. S5F).
This result is consistent with kap-1 phosphorylation promoting
release of kap-1/HP1/suv39h1 from DSBs. Intriguingly, the
phospho-mimic kap-1S824D was poorly retained at both sites of
laser damage (Fig. 5C) and at DSBs created by the p84-ZFN
(Fig. S5F). We conclude that the rapid release of kap-1/HP1/
suv39h1 from DSBs requires the ATM-dependent phosphorylation of serine 824 of kap-1.
Because many early responses to DNA damage require poly
(ADP-ribose) polymerase (PARP) family members, which catalyze synthesis of PAR chains on the chromatin at DSBs (33), we
examined whether the rapid recruitment of kap-1/HP1/suv39h1
to DSBs required PARP activity. The parp inhibitor olaparib
(PARPi) did not alter interaction between kap-1, HP1, and
suv39h1 (Fig. S5A). However, inhibition of PARP blocked the
rapid recruitment of suv39h1 (Fig. 6A) and kap-1 (Fig. S6B)
to sites of DNA damage. Chromatin PARylation after DNA
damage was not altered in cells lacking suv39h1 (Fig. S6A),

Fig. 3. Recruitment of suv39h1 to DSBs requires kap-1 and HP1. (A) U2OS cells were transfected with control siRNA or siRNA to kap-1. DNA damage was
created by using a laser, and cells were allowed to recover for 0, 15, or 30 min. Cells were costained with antibody to suv39h1 (red) or H2AX (green). (B) U2OS
cells were transfected with control siRNA or siRNA to suv39h1. DNA damage was created by using a laser, and cells were allowed to recover for 0, 15, or 30
min. Cells were costained with antibody to kap1 (green) or H2AX (red). (C) Quantitation of results in A and B. The percentage of H2AX laser stripes that
colocalized with either suv39h1 or kap-1 stripes were noted. Results are SD (n = 2560 cells). (D) 293T cells expressing nonspecific shRNA (control) or shRNA
to kap-1 or suv39h1 were transfected with vector (Vec) or p84-ZFN (ZFN), followed by ChIP using HP1 antibody and primers 1.5 kb to the right of the DSB.
Results are SD (n = 3). (E) 293T cells expressing nonspecific shRNA (control) or shRNA against kap-1 (shKap1) were transfected with p84-ZFN (ZFN) or vector
(Vec) followed by ChIP using H3K9me3 antibody and primers located 1.5 kb to the right of the DSB. Results are SD (n = 3).

demonstrating that PARylation is upstream of the kap-1/HP1/

suv39h1 complex. Furthermore, siRNA to parp1 also blocked
recruitment of suv39h1 (Fig. S6C), implying that parp1, rather
than other parp family members, is required for recruitment of
kap-1/HP1/suv39h1 to damaged chromatin. PARPi led to a significant reduction in H3K9me3 at DSBs (Fig. 6C), consistent with the
failure to recruit suv39h1 to DSBs in the presence of PARPi
(Fig. 6A) or parp1 siRNA (Fig. S6C). In addition, because
H3K9me3 is important for full activation of ATM (4), PARPi
also inhibited phosphorylation of kap-1 by ATM after DNA
damage (Fig. 6B). Finally, depletion of suv39h1 increased cell
sensitivity to PARPi (Fig. 6D), consistent with previous reports
that cells lacking ATM are sensitive to PARP inhibition (34, 35).
Discussion
We have shown that the suv39h1 methyltransferase is rapidly
recruited to DSBs, where it functions to create domains of
H3K9me3 adjacent to DSBs. Previous work demonstrated that

the repressive HP1 and kap-1 proteins were also recruited to

DSBs (4, 22, 23, 32, 36), although how these proteins contributed
to DSB repair was not clear. Kap-1, HP1 family members, and
suv39h1 can form large repressive complexes (2428). Here,
we show that loading of suv39h1, kap-1, and HP1 at DSBs was
interdependent, with loss of any one protein inhibiting recruitment of the other two. This finding is consistent with the
idea that kap-1, HP1, and suv39h1 are recruited to DSBs as a
single kap-1/HP1/suv39h1 complex. We propose a model in which
loading of the kap-1/HP1/suv39h1 complex at DSBs increases
H3K9me3 methylation on nucleosomes on either side of the
DSB. This promotes recruitment of additional kap-1/HP1/suv39h1
(through interaction of HP1s chromodomain with H3K9me3),
which then methylates H3K9 on nucleosomes further from the
DSB. This process leads to cycles of kap-1/HP1/suv39h1 loading
and H3K9 methylation, which catalyzes the spreading of
H3K9me3 (and kap-1/HP1/suv39h1) along the chromatin. This
model is similar to the spreading of heterochromatin, in which an

Fig. 4. HP1s chromodomain is required to recruit kap-1, HP1, and suv39h1 to DSBs. (A) U2OS cells expressing myc-suv39h1 (SuvWT) or catalytically inactive
myc-suv39h1 (SuvMD) were exposed to laser damage and stained with antibody to myc and H2AX or kap-1 and H2AX. Some cells were preincubated in
dimethyloxalylglycine (DMOG) (1 mM/1 h). (B) U2OS cells expressing nonspecific shRNA (shVec) or shRNA to suv39h1 (shSuv) were exposed to laser damage
and costained with antibodies to HP1 and H2AX or kap-1 and H2AX. Some cells were preincubated in DMOG (1 mM/1 h). (C) U2OS cells expressing vector,
HP1 (HA-HP1), or HP1 with the chromodomain deleted (HA-HP1CD) were exposed to laser damage and costained with antibodies for HA and H2AX or
suv39h1 and H2AX. Some cells were preincubated in DMOG (1 mM/1 h).

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Ayrapetov et al.

initial nucleation event positions HP1 complexes containing

H3K9 methyltransferases on the chromatin (20, 37). Subsequent
cycles of H3K9 methylation and loading of HP1 complexes result
in the spreading of heterochromatin along the chromatin (26, 38,
39). In this way, an initial nucleation event at DSBs can spread
H3K9me3 and kap-1/HP1/suv39h1 along the chromatin domains
flanking the DSB, leading to the rapid formation of repressive
chromatin at the DSB.
The initial nucleation event that recruits kap-1/HP1/suv39h1 to
DSBs required parp1. Several PARP family members are
recruited to DSBs where they rapidly create PAR chains. PAR
provides docking sites for several proteins implicated in DSB
repair, including macroH2A and the ALC1 and NuRD remodeling complexes (4044). However, neither kap-1 nor HP1 nor
suv39h1 contain conserved PAR binding motifs or undergo
changes in PARylation after DNA damage. Thus, whether the
kap-1/HP1/suv39h1 complex binds directly to PAR chains on the
chromatin, or whether the complex contains additional PAR-binding subunits, remains to be determined. Alternatively, PARylation
may alter nucleosome structure at DSBs, thereby facilitating H3K9
methylation by the kap-1/HP1/suv39h1 complex. In either
case, both parp1 activity and H3K9me3 spreading are required to stably, but transiently, load kap-1/HP1/suv39h1
onto the chromatin.
H3K9me3 is required for activation of the Tip60 acetyltransferase (4, 17). However, because H3K9me3 is primarily
located within silent, heterochromatic regions (20, 37, 38), this
requirement suggests that Tip60 activity during DNA repair may
be restricted to chromatin domains with a high density of
H3K9me2/3. Here, we demonstrate that transient loading of
kap-1/HP1/suv39h1 at DSBs provides a mechanism for rapidly
increasing H3K9me3 in open (euchromatin) domains that lack
preexisting H3K9me3. Furthermore, reducing H3K9me3 by targeting either suv39h1 or PARP activity inhibited Tip60s acetyltransferase activity and attenuated activation of ATM by DSBs.
This finding is consistent with published studies in which loss of
MRN (2), Tip60 (4, 6), or acute PARP inhibition (35) led to
defective activation of ATMs kinase activity. Increased H3K9
methylation by the kap-1/HP1/suv39h1 complex is therefore
critical for full activation of Tip60 and ATM and for the repair of
DSBs within open, euchromatic regions of the genome.
The recruitment of kap-1/HP1/suv39h1 and the increase in
H3K9me3 may reflect a need to temporarily stabilize and heterochromatinize DSBs in open regions. Other repressive complexes,
such as NuRD and histone deacetylases (HDACs) (4346), also
Ayrapetov et al.

CELL BIOLOGY

Fig. 5. Phosphorylation of kap-1 by ATM releases suv39h, kap-1, and HP1

from DSBs. (A and B) U2OS cells were preincubated with ATMi (100 M) or
solvent for 60 min. After laser microirradiation, cells were immediately fixed
(0 min) or allowed to recover for 15 min and then costained with antibodies
to suv39h1 and H2AX (A) or kap-1 and H2AX (B). (C) U2OS cells expressing
wild-type kap-1 (mycKap1wt), kap-1 with an alanine mutation in the ATM
phosphorylation site (mycKap1S824D), or kap-1 with a phospho-mimic in the
same site (mycKap1S824D) were exposed to laser microirradiation and mycKap1
and H2AX detected with myc or H2AX antibody.

transiently accumulate at DSBs, supporting this idea. The formation of repressive structures at DSBs in open chromatin has
parallels with DSB repair in heterochromatin. DSB repair in
heterochromatin requires the ATM-dependent phosphorylation
of kap-1 (30, 31), which releases the repressive CHD3 chromatin
remodeler (47) and promotes chromatin relaxation and facilitates DSB repair (31, 32, 47). Thus, immediately after DNA
damage, both euchromatin and heterochromatin domains have
similar structural organization, including high density of H3K9me3
and the presence of repressive complexes, such as kap-1, HP1,
methyltransferases, HDACs, and CHD3/CHD4 remodeling
ATPases (31, 32, 4347). This rapid, but temporary, formation of
repressive chromatin may inhibit local transcription, compact the
local chromatin structure, and rewrite the local epigenetic
landscape, stabilizing open chromatin structures and limiting
DSB mobility during the initial moments following DSB production. However, because repressive chromatin inhibits DSB
repair (30, 31, 47), it is important that these repressive structures
are rapidly dismantled. As the damage response unfolds, H3K9
methylation increases, leading to Tip60 activation and increased
ATM kinase activity. ATM then phosphorylates kap-1, releasing
the repressive kap-1/HP1/suv39h1 from the chromatin and thereby
providing a negative feedback loop that regulates both H3K9
methylation and loading of the kap-1/HP1/suv39h1 complex at
DSBs. In heterochromatin, kap-1 phosphorylation releases the
CHD3 complex (47), leading to relaxation of the chromatin structure (31), although kap-1 remains associated with the heterochromatin. The retention of phosphorylated kap-1 in heterochromatin
may result from the presence of Kruppel-associated box zinc finger proteins (21), which anchor kap-1 to heterochromatin, but
which are absent from open, euchromatin regions. In this way, the
compact structure of heterochromatin and the transient establishment of repressive chromatin at DSBs in open regions can be
reversed through a common mechanism dependent on the ATM
kinase. This structure then allows further chromatin processing to

Fig. 6. Chromatin PARylation recruits kap-1, HP1, and suv39h1 to DSBs. (A)
U2OS cells were preincubated with PARPi (olaparib; 1 h/20 M), followed by
laser microirradiation. Cells were either fixed (0 min) or allowed to recover
for 15 min, and then costained with antibody to suv39h1 and H2AX. (B)
U2OS cells were preincubated in PARPi (olaparib; 20 M) for 1 h, and then
exposed to bleomycin (5 M) for the indicated times. Kap1, pkap1, and tubulin were monitored by Western blot analysis. (C) 293T cells were transfected with vector (Vec) or p84-ZFN (ZFN), followed by solvent (control) or
PARPi (olaparib; 20M). ChIP was carried out by using H3K9me3 antibody
and primers located 1.5 kb to the right of the DSB. Results are SD (n = 3).
(D) 293T cells expressing nonspecific shRNA () or shRNA targeting suv39h1
() were incubated with PARPi for 24 h, and clonogenic cell survival assays
were carried out. Results are SE (n = 3).

PNAS | June 24, 2014 | vol. 111 | no. 25 | 9173

create open, flexible chromatin structures, which are essential for

DSB repair (11).
Dynamic methylation of histones and phosphorylation of kap1 during DSB repair therefore provide a regulated mechanism
for increasing compaction of open chromatin and decreasing the
compaction of heterochromatin, so that DSBs in both regions
begin to have similar epigenetic and structural organization. This
process is critical for establishing H3K9me3 for activation of
Tip60 and the ATM kinase, as well as for contributing to the early
processing of the chromatin at DSBs. These dynamic changes in
chromatin organization are therefore critical for creating a common chromatin template that is an efficient substrate for the HR
or NHEJ DSB repair pathways.
Materials and Methods
Details on cell growth, HR assays, transfection, antibodies, plasmid construction,
Western blot, mRNA analysis, and shRNA/siRNA are in SI Materials
and Methods.

agarose beads precoated with sperm DNA. After washing, immunopurified

chromatin was eluted and digested with proteinase K, and purified DNA was
quantified by quantitative RT-PCR (RT-qPCR) using the Step One Plus realtime PCR system (Applied Biosystems). Results are expressed as fold increase
in signal relative to uncut sample. Detailed protocols, primer pairs, and ChIP
grade antibodies are described in SI Materials and Methods.
Laser Microirradiation and Immunofluorescence. Laser damage was produced
by using a 30-mW, 405-nm diode laser focused through the 40-Plan Apochromat/1.25-N.A. oil objective (Leica TCS SP5; Leica Microsystems) in combination
with Hoechst 33258 (12). The time between the initial laser exposure and termination by fixation was 5 min, which is referred to as time 0. At least 50 nuclei
were microirradiated per slide. For recruitment of brca1 and RPA32 to ionizing
radiation induced foci, cells were cultured on coverslips and irradiated in a Cs137
irradiator. Cells were fixed and incubated with primary and secondary antibodies
as described in SI Materials and Methods and imaged by using a Zeiss AxioImager Z1 microscope.

ChIP. For ChIP assays (12, 13), cross-linked chromatin was sonicated, and
equivalent amounts were incubated with primary antibody and protein G

ACKNOWLEDGMENTS. We thank Sangamo Biosciences for p84-ZFN and

Dipanjan Chowdhury for the kap1 constructs. This work was supported by
National Institutes of Health Grants CA64585, CA93602, and CA177884 (to B.D.P.).

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