DNA double-strand breaks promote of H3 on 9 and transient formation of repressive

Marina K. Ayrapetova,1, Ozge Gursoy-Yuzugullua,1, Chang Xua,b,YeXua, and Brendan D. Pricea,2

aDepartment of Radiation Oncology, Dana–Farber 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, People’s 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) Dynamic changes in histone modification are critical for regulating acetylation and ubiquitination of the chromatin and loading of DNA double-strand break (DSB) repair. Activation of the Tip60 DNA repair proteins is therefore critical for DSB repair. acetyltransferase by DSBs requires interaction of Tip60 with his- Activation of Tip60’s acetyltransferase activity requires in- tone H3 methylated on lysine 9 (H3K9me3). However, how H3K9 teraction between Tip60’s chromodomain and meth- methylation is regulated during DSB repair is not known. Here, we ylated on lysine 9 (H3K9me3) on at the break (4, 6). demonstrate that a complex containing kap-1, HP1, and the H3K9 This interaction, in combination with tyrosine phosphorylation of ’ methyltransferase suv39h1 is rapidly loaded onto the chromatin at Tip60 (17), increases Tip60 s acetyltransferase activity and pro- – DSBs. Suv39h1 methylates H3K9, facilitating loading of additional motes acetylation of both the ATM kinase and histone H4 (4 6, kap-1/HP1/suv39h1 through binding of HP1’s chromodomain to 17). Consequently, loss of H3K9me2/3 leads to failure to activate the nascent H3K9me3. This process initiates cycles of kap-1/HP1/ the ATM signaling pathway, loss of H4 acetylation during DSB repair, disruption of , genomic instability, and suv39h1 loading and H3K9 methylation that facilitate spreading of defective DSB repair (4, 17–19). H3K9me3s therefore play a crit- H3K9me3 and kap-1/HP1/suv39h1 complexes for tens of kilobases ical role in linking chromatin structure at DSBs to the activation of away from the DSB. These domains of H3K9me3 function to acti- DSB signaling proteins such as Tip60 and ATM. vate the Tip60 acetyltransferase, allowing Tip60 to acetylate both How Tip60 gains access to H3K9me3 and how H3K9me3 levels ataxia telangiectasia-mutated (ATM) kinase and histone H4. Conse- at DSBs are regulated is not known. H3K9me3 is concentrated quently, cells lacking suv39h1 display defective activation of Tip60 in heterochromatin domains, where it recruits HP1, kap-1, and and ATM, decreased DSB repair, and increased radiosensitivity. Im- H3K9 methyltransferases (20, 21) to maintain the silent, compact portantly, activated ATM rapidly phosphorylates kap-1, leading to conformation of heterochromatin (20). This implies that Tip60’s release of the repressive kap-1/HP1/suv39h1 complex from the acetyltransferase activity can only be activated at DSBs in regions chromatin. ATM activation therefore functions as a negative feed- of high H3K9me3 density, such as heterochromatin. Alterna- back loop to remove repressive suv39h1 complexes at DSBs, which tively, H3K9 methylation may be actively increased at DSBs in may limit DSB repair. Recruitment of kap-1/HP1/suv39h1 to DSBs regions of low H3K9me3 density to allow for Tip60 activation and therefore provides a mechanism for transiently increasing the levels of H3K9me3 in open chromatin domains that lack H3K9me3 and Significance thereby promoting efficient activation of Tip60 and ATM in these regions. Further, transient formation of repressive chromatin may Double-strand break (DSB) repair initiates dynamic changes in be critical for stabilizing the damaged chromatin and for remodel- histone modifications that are required to maintain genome ing the chromatin to create an efficient template for the DNA stability. Methylation of histone H3 lysine-9 (H3K9me3) is repair machinery. critical for activating ataxia telangiectasia-mutated (ATM) ki- CELL BIOLOGY nase, but how H3K9 methylation is regulated at DSBs is un- | homologous recombination known. We show that a complex containing the suv39h1 methyltransferase is rapidly recruited to DSBs, where it directs NA double-strand breaks (DSBs) are toxic and must be H3K9 methylation on large chromatin domains adjacent to the Drepaired to maintain genomic stability. Detection of DSBs DSB. This process results in transient formation of repressive requires recruitment of the mre11–rad50–nbs1 (MRN) complex chromatin and serves to both stabilize the chromatin structure to the DNA ends (1). MRN then recruits and activates the ataxia and promote activation of DSB-signaling proteins, including ATM telangiectasia-mutated (ATM) kinase (2, 3) through a mecha- kinase. Dynamic changes in H3K9 modification in euchro- nism that also requires the Tip60 acetyltransferase (3). Tip60 matin by suv39h1 are therefore one of the earliest signaling directly acetylates and activates ATM’s kinase activity (4–6) and events required for processing and remodeling of the damaged functions, in combination with MRN, to promote ATM-dependent chromatin template. phosphorylation of DSB repair proteins (3), including histone H2AX. This process creates domains of phosphorylated H2AX Author contributions: O.G.-Y. and B.D.P. designed research; B.D.P. conceived and planned γ the study, with input from M.K.A. and O.G.-Y.; M.K.A., O.G.-Y., C.X., and Y.X. performed ( H2AX) extending for hundreds of kilobases along the chro- research; M.K.A. performed laser microirradiation, plasmid construction, and cell-based matin (7, 8). Mdc1 then binds to γH2AX, providing a landing analysis; O.G.-Y. performed the ChIP experiments with contributions from C.X.; Y.X. con- pad for other DSB repair proteins, including the RNF8/RNF168 tributed to assay development; M.K.A., O.G.-Y., and B.D.P. analyzed data; and M.K.A., ubiquitin ligases (1, 3, 9, 10). Tip60 also plays a critical role in O.G.-Y., and B.D.P. wrote the paper. regulating chromatin structure at DSBs as part of the NuA4– The authors declare no conflict of interest. Tip60 complex (11). NuA4-Tip60 catalyzes histone exchange (via This article is a PNAS Direct Submission. the p400 ATPase subunit) and acetylation of histone H4 (by Tip60) Freely available online through the PNAS open access option. at DSBs (12–15), leading to the formation of open, flexible chro- 1M.K.A. and O.G.-Y. contributed equally to this work. matin domains adjacent to the break (12, 13). These open chro- 2To whom correspondence should be addressed. E-mail: [email protected]. matin structures then facilitate histone ubiquitination, the loading This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. of brca1 and 53BP1, and repair of the DSB (13, 16). The ordered 1073/pnas.1403565111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1403565111 PNAS | June 24, 2014 | vol. 111 | no. 25 | 9169–9174 Downloaded by guest on September 23, 2021 efficient DSB repair in euchromatin. Understanding the dy- namics of H3K9 methylation at DSBs is therefore critical to un- derstanding 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, acti- vating Tip60’s acetyltransferase activity and promoting the sub- sequent 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 com- plex is transient, and the complex is rapidly released from the chromatin through a negative feedback loop driven by ATM- dependent 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 p84–zinc 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/). Chro- matin IP (ChIP) demonstrated increased phosphorylation of H2AX (γH2AX) 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 in- crease 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 or H4K20me2 was seen at the DSB (Fig. S1B). Fi- nally, H3K9me3 was not increased at a distal chromosome site (Fig. S1C), indicating that the increased H3K9me3 is re- stricted 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. Fig. 1. Suv39h1 promotes H3K9 methylation at DSBs. (A) 293T cells trans- Interaction between Tip60 and H3K9me3 (4) promotes acet- fected with p84-ZFN were processed for ChIP by using γH2AX or ylation of ATM (5, 6, 17) and histone H4 (12–14) by Tip60. H3K9me3 antibodies, followed by quantitative RT-PCR (RT-qPCR) with Depletion of suv39h1 reduced inducible H3K9me3 (Fig. 1C) and primer pairs located at the indicated positions. Results are fold enrichment inhibited acetylation of histone H4 by Tip60 (Fig. 2A). Fur- relative to uncut DNA, which is assigned a value of 1 (solid black line; Uncut). thermore, depletion of suv39h1 attenuated ATM activation Results are ±SD (n = 3). (B) 293T cells transfected with p84-ZFN (ZFN) or (Fig. S2A) and reduced phosphorylation of kap-1 (Fig. S2B). vector (Vec) were processed for ChIP by using antibodies to H3, γH2AX, This finding is consistent with methylation of H3K9 by suv39h1 H3K9me3, or H3K9me2 and primer pairs located 1.5 kb to the right of the playing an essential role in activation of Tip60’s acetyltransferase DSB. Results are expressed as fold enrichment relative to uncut DNA. Results activity and the subsequent acetylation and activation of the are ±SD (n = 3). (C) 293T cells were transfected with nonspecific (−) or siRNA ATM kinase. Furthermore, cells lacking suv39h1 have increased targeting suv39h1 (+). Forty-eight hours later, cells were transfected with radiation sensitivity (Fig. 2B) and reduced homologous recombi- vector (Vec) or p84-ZFN (ZFN) and processed for ChIP by using antibody C against H3K9me3 and primers located 1.5 kb to either side of the DSB. nation (HR)-mediated repair (Fig. 2 ). Finally, recruitment of ± = brca1 and RPA32 to DSBs (Fig. 2D) were reduced following loss 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 of suv39h1, consistent with the decrease in HR-mediated repair ± = C located 1.5 kb to either side of the DSB. Results are SD (n 3). (E)U2OS in suv39h1-deficient cells (Fig. 2 ). However, nonhomologous cells were transfected with nonspecific (control) or suv39h1-specific end-joining (NHEJ) activity was not significantly altered by loss (siSuv39h1) siRNA. Forty-eight hours later, focused regions of DNA damage of suv39h1 (Fig. S2C), implying that regulation of the Ku70/80 were produced by using a scanning laser system. Cells were fixed for im- complex and DNA–PKcs activity does not require H3K9 methyla- munofluorescent staining by using antibodies to γH2AX (green) or suv39h1 tion. These results are consistent with previous studies demonstrating (red). Nuclei were stained with DAPI (blue). 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 acti- kap-1 interacts with the HP1α,HP1β, and suv39h1 and that this vating Tip60, controlling ATM signaling, and in directing DSB interaction was not altered by DNA damage (Fig. S3 B and C). repair and maintaining genomic stability. When laser “striping” was used to create focused regions of DNA Two heterochromatin-binding proteins, kap-1 and HP1, are damage, suv39h1 (Fig. 3A) and kap-1 (Fig. 3B) were recruited to corecruited to sites of DNA damage (22, 23), although how they sites of DNA damage with similar kinetics, such that ∼90% of impact DSB repair is not clear. However, suv39h1 can interact with γH2AX stripes were colocalized with suv39h1 and kap-1 (Fig. kap-1/HP1 repressor complexes (20, 21, 24), suggesting that kap-1 3C). Furthermore, ChIP demonstrated that HP1β was also and HP1 may recruit suv39h1 to DSBs. Initially, we confirmed that recruited to sites of DNA damage (Fig. 3D). Importantly,

9170 | www.pnas.org/cgi/doi/10.1073/pnas.1403565111 Ayrapetov et al. Downloaded by guest on September 23, 2021 laser damage, and, furthermore, it blocked recruitment of kap-1 (Fig. 4A), such that <5% of γH2AX stripes in suv39h1MD cells contained either kap-1 or suv39h1 (see Fig. S4E for quantita- tion). 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 γH2AX stripes in suv39h1MD cells then colocalized with suv39h1 and kap-1 (Fig. S4E). Further, DMOG rescued recruitment of kap-1 and HP1α, 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 HP1α binds to H3K9me3, we deleted HP1α’s chromodomain (Fig. S4D). Loss of HP1α’s chromodomain did not alter in- teraction of HP1α with either suv39h1 or kap-1 (Fig. S4 F and G). Importantly, HP1αCD was not recruited to sites of laser micro- irradiation and inhibited corecruitment of suv39h1 (Fig. 4C). Furthermore, increasing H3K9me3 with DMOG did not restore loading of HP1 or suv39h1 in HP1αCD cells (Fig. 4C), demon- strating that both HP1α’s chromodomain and H3K9me3 are re- quired 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 HP1’s chromo- domain 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. Fig. 2. Suv39h1 regulates Tip60 activity, genomic stability, and homologous Kap-1, HP1, and suv39h1 are only retained at the DSB during recombination (HR)-mediated repair. (A) 293T cells expressing nonspecific the first few minutes after damage (Figs. 3 and 4). Because the shRNA (control) or shRNA targeting suv39h1 were transfected with vector ATM-dependent phosphorylation of kap-1 weakens kap-1’s in- (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. teraction with chromatin (30, 31), we examined whether ATM Results are ±SD (n = 3). (B) 293T cells expressing nonspecific shRNA (●)or was required to release kap-1/HP1/suv39h1 from DSBs. The ○ ATM inhibitor Ku-55933 (ATMi) did not alter interaction between shRNA targeting suv39h1 ( ) were irradiated, and clonogenic cell survival A A assays were carried out. Results are ±SD (n = 3). (C) U2OS cells containing kap-1 and suv39h1 (Fig. S5 ). Suv39h1 (Fig. 5 )andkap-1(Fig. aGFP–HR reporter were stably transfected with nonspecific (NS) or shRNA 5B) were recruited to DSBs in the presence of ATMi. However, targeting suv39h1, followed by transient transfection with vector (Vec) or ATMi blocked release of suv39h1 and kap-1 (Fig. 5 A and B) and the I-Sce1 endonuclease. GFP-positive cells were counted by FACS. Results increased retention of kap-1 at p84-ZFN–generated DSBs (Fig. are ±SD (n = 4 biological replicates). (D) U2OS cells expressing nonspecific (−) S5B). Similarly, inactivation of the MRN complex, which is re- CELL BIOLOGY or shRNA targeting suv39h1 (+) were irradiated (2 Gy), allowed to recover quired for ATM activation (2), blocked phosphorylation of kap-1 for 30 min, and stained with antibodies to γH2AX, brca1, or RPA32. Cells by ATM (Fig. S5D) and prevented release of suv39h1 from sites with more than five foci were scored as positive. Results are ±SD (n = 3). P of DNA damage (Fig. S5C). Phosphorylation of serine 824 of value was determined by using a t test. kap-1 by ATM weakens kap-1 interaction with chromatin (30–32). Kap-1wt,kap-1S824A, and kap-1S824D (containing a phospho-mimic) were expressed in U2OS cells (Fig. S5E). Kap-1wt was transiently suv39h1, kap-1, and HP1 were only transiently retained at damage recruited to and released from regions of DNA damage, whereas A B sites (Fig. 3 and ), indicating that they function during the the nonphosphorylatable kap-1S824A was recruited to DSBs but initial few minutes following DSB production. Furthermore, de- retained at DSBs for an extended time (Fig. 5C and Fig. S5F). A pletion of kap-1 (Fig. S3 ) blocked recruitment of both suv39h1 This result is consistent with kap-1 phosphorylation promoting A C β D (Fig. 3 and ) and HP1 (Fig. 3 ) to DSBs. Similarly, de- release of kap-1/HP1/suv39h1 from DSBs. Intriguingly, the pletion of suv39h1 blocked recruitment of kap-1 (Fig. 3 B and C phospho-mimic kap-1S824D was poorly retained at both sites of and Fig. S3D) and HP1β (Fig. 3D) to DSBs. Loss of any one of laser damage (Fig. 5C) and at DSBs created by the p84-ZFN suv39h1, kap-1, or HP1 therefore prevents loading of all three (Fig. S5F). We conclude that the rapid release of kap-1/HP1/ proteins at the DSB. Finally, depletion of kap-1, which blocks suv39h1 from DSBs requires the ATM-dependent phosphoryla- recruitment of both suv39h1 and HP1 to DSBs, abolished the tion of serine 824 of kap-1. increase in H3K9me3 at DSBs (Fig. 3E). Combining these data Because many early responses to DNA damage require poly with previous work demonstrating direct interaction between (ADP-ribose) polymerase (PARP) family members, which cata- kap-1, HP1 family members, and suv39h1 (24–28) implies that lyze synthesis of PAR chains on the chromatin at DSBs (33), we kap-1, HP1, and suv39h1 are recruited to DSBs as a single kap-1/ examined whether the rapid recruitment of kap-1/HP1/suv39h1 HP1/suv39h1 complex and that it is this complex that directs to DSBs required PARP activity. The parp inhibitor olaparib H3K9 methylation at DSBs. (PARPi) did not alter interaction between kap-1, HP1α, and Next, we examined suv39h1’s methyltransferase activity. suv39h1 (Fig. S5A). However, inhibition of PARP blocked the Suv39h1MD, containing a point mutation in the catalytic domain, rapid recruitment of suv39h1 (Fig. 6A) and kap-1 (Fig. S6B) was efficiently incorporated into the kap-1/HP1/suv39h1 complex to sites of DNA damage. Chromatin PARylation after DNA (Fig. S4A). However, suv39h1MD was not recruited to regions of damage was not altered in cells lacking suv39h1 (Fig. S6A),

Ayrapetov et al. PNAS | June 24, 2014 | vol. 111 | no. 25 | 9171 Downloaded by guest on September 23, 2021 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 γH2AX (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 γH2AX (red). (C) Quantitation of results in A and B. The percentage of γH2AX laser stripes that colocalized with either suv39h1 or kap-1 stripes were noted. Results are ±SD (n = 25–60 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 HP1β 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/HP1α/ the repressive HP1 and kap-1 proteins were also recruited to suv39h1 complex. Furthermore, siRNA to parp1 also blocked DSBs (4, 22, 23, 32, 36), although how these proteins contributed recruitment of suv39h1 (Fig. S6C), implying that parp1, rather to DSB repair was not clear. Kap-1, HP1 family members, and than other parp family members, is required for recruitment of suv39h1 can form large repressive complexes (24–28). Here, kap-1/HP1/suv39h1 to damaged chromatin. PARPi led to a signifi- we show that loading of suv39h1, kap-1, and HP1 at DSBs was cant reduction in H3K9me3 at DSBs (Fig. 6C), consistent with the interdependent, with loss of any one protein inhibiting re- failure to recruit suv39h1 to DSBs in the presence of PARPi cruitment of the other two. This finding is consistent with the (Fig. 6A) or parp1 siRNA (Fig. S6C). In addition, because idea that kap-1, HP1, and suv39h1 are recruited to DSBs as a H3K9me3 is important for full activation of ATM (4), PARPi single kap-1/HP1/suv39h1 complex. We propose a model in which also inhibited phosphorylation of kap-1 by ATM after DNA loading of the kap-1/HP1/suv39h1 complex at DSBs increases damage (Fig. 6B). Finally, depletion of suv39h1 increased cell H3K9me3 methylation on nucleosomes on either side of the sensitivity to PARPi (Fig. 6D), consistent with previous reports DSB. This promotes recruitment of additional kap-1/HP1/suv39h1 that cells lacking ATM are sensitive to PARP inhibition (34, 35). (through interaction of HP1’s chromodomain with H3K9me3), which then methylates H3K9 on nucleosomes further from the Discussion DSB. This process leads to cycles of kap-1/HP1/suv39h1 loading We have shown that the suv39h1 methyltransferase is rapidly and H3K9 methylation, which catalyzes the spreading of recruited to DSBs, where it functions to create domains of H3K9me3 (and kap-1/HP1/suv39h1) along the chromatin. This H3K9me3 adjacent to DSBs. Previous work demonstrated that model is similar to the spreading of heterochromatin, in which an

Fig. 4. HP1’s 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 γH2AX or kap-1 and γH2AX. 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 HP1α and γH2AX or kap-1 and γH2AX. Some cells were preincubated in DMOG (1 mM/1 h). (C) U2OS cells expressing vector, HP1α (HA-HP1α), or HP1α with the chromodomain deleted (HA-HP1αCD) were exposed to laser damage and costained with antibodies for HA and γH2AX or suv39h1 and γH2AX. Some cells were preincubated in DMOG (1 mM/1 h).

9172 | www.pnas.org/cgi/doi/10.1073/pnas.1403565111 Ayrapetov et al. Downloaded by guest on September 23, 2021 transiently accumulate at DSBs, supporting this idea. The for- mation 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 facili- tates 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, HP1α, methyltransferases, HDACs, and CHD3/CHD4 remodeling ATPases (31, 32, 43–47). 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 pro- Fig. 5. Phosphorylation of kap-1 by ATM releases suv39h, kap-1, and HP1 duction. However, because repressive chromatin inhibits DSB μ from DSBs. (A and B) U2OS cells were preincubated with ATMi (100 M) or repair (30, 31, 47), it is important that these repressive structures solvent for 60 min. After laser microirradiation, cells were immediately fixed are rapidly dismantled. As the damage response unfolds, H3K9 (0 min) or allowed to recover for 15 min and then costained with antibodies to suv39h1 and γH2AX (A) or kap-1 and γH2AX (B). (C) U2OS cells expressing methylation increases, leading to Tip60 activation and increased wild-type kap-1 (mycKap1wt), kap-1 with an alanine mutation in the ATM ATM kinase activity. ATM then phosphorylates kap-1, releasing phosphorylation site (mycKap1S824D), or kap-1 with a phospho-mimic in the the repressive kap-1/HP1/suv39h1 from the chromatin and thereby same site (mycKap1S824D) were exposed to laser microirradiation and mycKap1 providing a negative feedback loop that regulates both H3K9 and γH2AX detected with myc or γH2AX antibody. 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 struc- initial nucleation event positions HP1 complexes containing ture (31), although kap-1 remains associated with the heterochro- H3K9 methyltransferases on the chromatin (20, 37). Subsequent matin. The retention of phosphorylated kap-1 in heterochromatin cycles of H3K9 methylation and loading of HP1 complexes result may result from the presence of Kruppel-associated box zinc fin- in the spreading of heterochromatin along the chromatin (26, 38, ger proteins (21), which anchor kap-1 to heterochromatin, but 39). In this way, an initial nucleation event at DSBs can spread which are absent from open, euchromatin regions. In this way, the H3K9me3 and kap-1/HP1/suv39h1 along the chromatin domains compact structure of heterochromatin and the transient estab- flanking the DSB, leading to the rapid formation of repressive lishment of repressive chromatin at DSBs in open regions can be chromatin at the DSB. reversed through a common mechanism dependent on the ATM The initial nucleation event that recruits kap-1/HP1/suv39h1 to kinase. This structure then allows further chromatin processing 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 remod- eling complexes (40–44). 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/HP1α/suv39h1 complex binds directly to PAR chains on the chromatin, or whether the complex contains additional PAR-bind-

ing subunits, remains to be determined. Alternatively, PARylation CELL BIOLOGY may alter structure at DSBs, thereby facilitating H3K9 methylation by the kap-1/HP1/suv39h1 complex. In either case, both parp1 activity and H3K9me3 spreading are re- quired to stably, but transiently, load kap-1/HP1α/suv39h1 onto the chromatin. H3K9me3 is required for activation of the Tip60 acetyl- transferase (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 tar- ’ Fig. 6. Chromatin PARylation recruits kap-1, HP1, and suv39h1 to DSBs. (A) geting either suv39h1 or PARP activity inhibited Tip60 s acetyl- μ transferase activity and attenuated activation of ATM by DSBs. 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 This finding is consistent with published studies in which loss of for 15 min, and then costained with antibody to suv39h1 and γH2AX. (B) MRN (2), Tip60 (4, 6), or acute PARP inhibition (35) led to μ ’ U2OS cells were preincubated in PARPi (olaparib; 20 M) for 1 h, and then defective activation of ATM s kinase activity. Increased H3K9 exposed to bleomycin (5 μM) for the indicated times. Kap1, pkap1, and tu- methylation by the kap-1/HP1/suv39h1 complex is therefore bulin were monitored by Western blot analysis. (C) 293T cells were trans- critical for full activation of Tip60 and ATM and for the repair of fected with vector (Vec) or p84-ZFN (ZFN), followed by solvent (control) or DSBs within open, euchromatic regions of the genome. PARPi (olaparib; 20μM). ChIP was carried out by using H3K9me3 antibody The recruitment of kap-1/HP1/suv39h1 and the increase in and primers located 1.5 kb to the right of the DSB. Results are ±SD (n = 3). H3K9me3 may reflect a need to temporarily stabilize and “heter- (D) 293T cells expressing nonspecific shRNA (○) or shRNA targeting suv39h1 ochromatinize” DSBs in open regions. Other repressive complexes, (●) were incubated with PARPi for 24 h, and clonogenic cell survival assays such as NuRD and histone deacetylases (HDACs) (43–46), also were carried out. Results are ±SE (n = 3).

Ayrapetov et al. PNAS | June 24, 2014 | vol. 111 | no. 25 | 9173 Downloaded by guest on September 23, 2021 create open, flexible chromatin structures, which are essential for agarose beads precoated with sperm DNA. After washing, immunopurified DSB repair (11). chromatin was eluted and digested with proteinase K, and purified DNA was Dynamic methylation of and phosphorylation of kap- quantified by quantitative RT-PCR (RT-qPCR) using the Step One Plus real- 1 during DSB repair therefore provide a regulated mechanism time PCR system (Applied Biosystems). Results are expressed as fold increase for increasing compaction of open chromatin and decreasing the in signal relative to uncut sample. Detailed protocols, primer pairs, and ChIP compaction of heterochromatin, so that DSBs in both regions grade antibodies are described in SI Materials and Methods. begin to have similar epigenetic and structural organization. This process is critical for establishing H3K9me3 for activation of Laser Microirradiation and Immunofluorescence. Laser damage was produced Tip60 and the ATM kinase, as well as for contributing to the early by using a 30-mW, 405-nm diode laser focused through the 40×-Plan Apochro- processing of the chromatin at DSBs. These dynamic changes in mat/1.25-N.A. oil objective (Leica TCS SP5; Leica Microsystems) in combination chromatin organization are therefore critical for creating a com- with Hoechst 33258 (12). The time between the initial laser exposure and ter- mon chromatin template that is an efficient substrate for the HR mination by fixation was 5 min, which is referred to as time 0. At least 50 nuclei or NHEJ DSB repair pathways. 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 Materials and Methods irradiator. Cells were fixed and incubated with primary and secondary antibodies Details on cell growth, HR assays, transfection, antibodies, plasmid construction, as described in SI Materials and Methods and imaged by using a Zeiss AxioIm- Western blot, mRNA analysis, and shRNA/siRNA are in SI Materials ager Z1 microscope. and Methods. ACKNOWLEDGMENTS. We thank Sangamo Biosciences for p84-ZFN and ChIP. For ChIP assays (12, 13), cross-linked chromatin was sonicated, and Dipanjan Chowdhury for the kap1 constructs. This work was supported by equivalent amounts were incubated with primary antibody and protein G National Institutes of Health Grants CA64585, CA93602, and CA177884 (to B.D.P.).

1. Ciccia A, Elledge SJ (2010) The DNA damage response: Making it safe to play with 26. Nielsen AL, et al. (1999) Interaction with members of the heterochromatin protein 1 knives. Mol Cell 40(2):179–204. (HP1) family and histone deacetylation are differentially involved in transcriptional 2. Shiloh Y, Ziv Y (2013) The ATM protein kinase: Regulating the cellular response to silencing by members of the TIF1 family. EMBO J 18(22):6385–6395. genotoxic stress, and more. Nat Rev Mol Cell Biol 14(4):197–210. 27. Li X, et al. (2010) SUMOylation of the transcriptional co-repressor KAP1 is regulated 3. Sun Y, Jiang X, Price BD (2010) Tip60: Connecting chromatin to DNA damage sig- by the serine and threonine phosphatase PP1. Sci Signal 3(119):ra32. naling. Cell Cycle 9(5):930–936. 28. Ivanov AV, et al. (2007) PHD domain-mediated E3 ligase activity directs intramolecular 4. Sun Y, et al. (2009) Histone H3 methylation links DNA damage detection to activation sumoylation of an adjacent bromodomain required for gene silencing. Mol Cell 28(5): – of the tumour suppressor Tip60. Nat Cell Biol 11(11):1376–1382. 823 837. α 5. Sun Y, Xu Y, Roy K, Price BD (2007) DNA damage-induced acetylation of lysine 3016 of 29. Ayrapetov MK, et al. (2011) Activation of Hif1 by the prolylhydroxylase inhibitor ATM activates ATM kinase activity. Mol Cell Biol 27(24):8502–8509. dimethyoxalyglycine decreases radiosensitivity. PLoS ONE 6(10):e26064. 6. Sun Y, Jiang X, Chen S, Fernandes N, Price BD (2005) A role for the Tip60 histone 30. Noon AT, et al. (2010) 53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair. Nat Cell Biol 12(2): acetyltransferase in the acetylation and activation of ATM. Proc Natl Acad Sci USA 177–184. 102(37):13182–13187. 31. Goodarzi AA, et al. (2008) ATM signaling facilitates repair of DNA double-strand 7. Rogakou EP, Boon C, Redon C, Bonner WM (1999) Megabase chromatin domains breaks associated with heterochromatin. Mol Cell 31(2):167–177. involved in DNA double-strand breaks in vivo. J Cell Biol 146(5):905–916. 32. Ziv Y, et al. (2006) Chromatin relaxation in response to DNA double-strand breaks is 8. Iacovoni JS, et al. (2010) High-resolution profiling of gammaH2AX around DNA modulated by a novel ATM- and KAP-1 dependent pathway. Nat Cell Biol 8(8): – double strand breaks in the mammalian genome. EMBO J 29(8):1446 1457. 870–876. 9. Jackson SP, Durocher D (2013) Regulation of DNA damage responses by ubiquitin and 33. Gibson BA, Kraus WL (2012) New insights into the molecular and cellular functions of – SUMO. Mol Cell 49(5):795 807. poly(ADP-ribose) and PARPs. Nat Rev Mol Cell Biol 13(7):411–424. 10. Fradet-Turcotte A, et al. (2013) 53BP1 is a reader of the DNA-damage-induced H2A 34. Williamson CT, et al. (2010) ATM deficiency sensitizes mantle cell lymphoma cells to Lys 15 ubiquitin mark. Nature 499(7456):50–54. poly(ADP-ribose) polymerase-1 inhibitors. Mol Cancer Ther 9(2):347–357. 11. Price BD, D’Andrea AD (2013) Chromatin remodeling at DNA double-strand breaks. 35. Haince JF, et al. (2007) Ataxia telangiectasia mutated (ATM) signaling network is Cell 152(6):1344–1354. modulated by a novel poly(ADP-ribose)-dependent pathway in the early response to 12. Xu Y, et al. (2012) Histone H2A.Z controls a critical chromatin remodeling step re- DNA-damaging agents. J Biol Chem 282(22):16441–16453. quired for DNA double-strand break repair. Mol Cell 48(5):723–733. 36. Ayoub N, Jeyasekharan AD, Bernal JA, Venkitaraman AR (2008) HP1-beta mobiliza- 13. Xu Y, et al. (2010) The p400 ATPase regulates nucleosome stability and chromatin tion promotes chromatin changes that initiate the DNA damage response. Nature ubiquitination during DNA repair. J Cell Biol 191(1):31–43. 453(7195):682–686. 14. Murr R, et al. (2006) Histone acetylation by Trrap-Tip60 modulates loading of repair 37. Tamaru H (2010) Confining euchromatin/heterochromatin territory: Jumonji crosses proteins and repair of DNA double-strand breaks. Nat Cell Biol 8(1):91–99. the line. Genes Dev 24(14):1465–1478. 15. Soria G, Polo SE, Almouzni G (2012) Prime, repair, restore: The active role of chro- 38. Hathaway NA, et al. (2012) Dynamics and memory of heterochromatin in living cells. matin in the DNA damage response. Mol Cell 46(6):722–734. Cell 149(7):1447–1460. 16. Tang J, et al. (2013) Acetylation limits 53BP1 association with damaged chromatin to 39. Cheutin T, et al. (2003) Maintenance of stable heterochromatin domains by dynamic – promote homologous recombination. Nat Struct Mol Biol 20(3):317–325. HP1 binding. Science 299(5607):721 725. 17. Kaidi A, Jackson SP (2013) KAT5 tyrosine phosphorylation couples chromatin sensing 40. Ahel D, et al. (2009) Poly(ADP-ribose)-dependent regulation of DNA repair by the – to ATM signalling. Nature 498(7452):70–74. chromatin remodeling enzyme ALC1. Science 325(5945):1240 1243. 18. Peters AH, et al. (2001) Loss of the Suv39h histone methyltransferases impairs 41. Xu C, Xu Y, Gursoy-Yuzugullu O, Price BD (2012) The histone variant macroH2A1.1 is recruited to DSBs through a mechanism involving PARP1. FEBS Lett 586(21): mammalian heterochromatin and genome stability. Cell 107(3):323–337. 3920–3925. 19. Peng JC, Karpen GH (2009) Heterochromatic genome stability requires regulators of 42. Timinszky G, et al. (2009) A macrodomain-containing histone rearranges chromatin histone H3 K9 methylation. PLoS Genet 5(3):e1000435. upon sensing PARP1 activation. Nat Struct Mol Biol 16(9):923–929. 20. Grewal SI, Jia S (2007) Heterochromatin revisited. Nat Rev Genet 8(1):35–46. 43. Polo SE, Kaidi A, Baskcomb L, Galanty Y, Jackson SP (2010) Regulation of DNA- 21. Iyengar S, Farnham PJ (2011) KAP1 protein: An enigmatic master regulator of the damage responses and cell-cycle progression by the chromatin remodelling factor genome. J Biol Chem 286(30):26267–26276. CHD4. EMBO J 29(18):3130–3139. 22. Baldeyron C, Soria G, Roche D, Cook AJ, Almouzni G (2011) HP1alpha recruitment to 44. Chou DM, et al. (2010) A chromatin localization screen reveals poly (ADP ribose)- DNA damage by p150CAF-1 promotes homologous recombination repair. J Cell Biol regulated recruitment of the repressive polycomb and NuRD complexes to sites of – 193(1):81 95. DNA damage. Proc Natl Acad Sci USA 107(43):18475–18480. 23. Luijsterburg MS, et al. (2009) Heterochromatin protein 1 is recruited to various types 45. Smeenk G, et al. (2010) The NuRD chromatin-remodeling complex regulates signaling – of DNA damage. J Cell Biol 185(4):577 586. and repair of DNA damage. J Cell Biol 190(5):741–749. 24. Fritsch L, et al. (2010) A subset of the histone H3 lysine 9 methyltransferases Suv39h1, 46. Miller KM, et al. (2010) Human HDAC1 and HDAC2 function in the DNA-damage response G9a, GLP, and SETDB1 participate in a multimeric complex. Mol Cell 37(1):46–56. to promote DNA nonhomologous end-joining. Nat Struct Mol Biol 17(9):1144–1151. 25. Lechner MS, Begg GE, Speicher DW, Rauscher FJ, 3rd (2000) Molecular determinants 47. Goodarzi AA, Kurka T, Jeggo PA (2011) KAP-1 phosphorylation regulates CHD3 nu- for targeting heterochromatin protein 1-mediated gene silencing: Direct chromoshadow cleosome remodeling during the DNA double-strand break response. Nat Struct domain-KAP-1 corepressor interaction is essential. MolCellBiol20(17):6449–6465. Mol Biol 18(7):831–839.

9174 | www.pnas.org/cgi/doi/10.1073/pnas.1403565111 Ayrapetov et al. Downloaded by guest on September 23, 2021