DNA double-strand breaks promote methylation of histone H3 on lysine 9 and transient formation of repressive chromatin.
ABSTRACT: 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 HP1's 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.
Project description:DNA double-strand break (DSB) repair involves complex interactions between chromatin and repair proteins, including Tip60, a tumour suppressor. Tip60 is an acetyltransferase that acetylates both histones and ATM (ataxia telangiectasia mutated) kinase. Inactivation of Tip60 leads to defective DNA repair and increased cancer risk. However, how DNA damage activates the acetyltransferase activity of Tip60 is not known. Here, we show that direct interaction between the chromodomain of Tip60 and histone H3 trimethylated on lysine 9 (H3K9me3) at DSBs activates the acetyltransferase activity of Tip60. Depletion of intracellular H3K9me3 blocks activation of the acetyltransferase activity of Tip60, resulting in defective ATM activation and widespread defects in DSB repair. In addition, the ability of Tip60 to access H3K9me3 is dependent on the DNA damage-induced displacement of HP1beta (heterochromatin protein 1beta) from H3K9me3. Finally, we demonstrate that the Mre11-Rad50-Nbs1 (MRN) complex targets Tip60 to H3K9me3, and is required to activate the acetyltransferase activity of Tip60. These results reveal a new function for H3K9me3 in coordinating activation of Tip60-dependent DNA repair pathways, and imply that aberrant patterns of histone methylation may contribute to cancer by altering the efficiency of DSB repair.
Project description:Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after ?-irradiation (?-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and ?H2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-? and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.
Project description:Radiation therapy is widely used to treat human malignancies, but many tumor types, including gliomas, exhibit significant radioresistance. Radiation therapy creates DNA double-strand breaks (DSBs), and DSB repair is linked to rapid changes in epigenetic modifications, including increased histone methylation. This increased histone methylation recruits DNA repair proteins which can then alter the local chromatin structure and promote repair. Consequently, combining inhibitors of specific histone methyltransferases with radiation therapy may increase tumor radiosensitivity, particularly in tumors with significant therapeutic resistance. Here, we demonstrate that inhibitors of the H4K20 methyltransferase SETD8 (UNC-0379) and the H3K9 methyltransferase G9a (BIX-01294) are effective radiosensitizers of human glioma cells. UNC-0379 blocked H4K20 methylation and reduced recruitment of the 53BP1 protein to DSBs, although this loss of 53BP1 caused only limited changes in radiosensitivity. In contrast, loss of H3K9 methylation through G9a inhibition with BIX-01294 increased radiosensitivity of a panel of glioma cells (SER2Gy range: 1.5 - 2.9). Further, loss of H3K9 methylation reduced DSB signaling dependent on H3K9, including reduced activation of the Tip60 acetyltransferase, loss of ATM signaling and reduced phosphorylation of the KAP-1 repressor. In addition, BIX-0194 inhibited DSB repair through both the homologous recombination and nonhomologous end-joining pathways. Inhibition of G9a and loss of H3K9 methylation is therefore an effective approach for increasing radiosensitivity of glioma cells. These results suggest that combining inhibitors of histone methyltransferases which are critical for DSB repair with radiation therapy may provide a new therapeutic route for sensitizing gliomas and other tumors to radiation therapy.
Project description:Non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the two main pathways for repairing DNA double-strand breaks (DSBs). During the G2 phase of the mammalian cell cycle, both processes can operate and chromatin structure is one important factor which determines DSB repair pathway choice. ATM facilitates the repair of heterochromatic DSBs by phosphorylating and inactivating the heterochromatin building factor KAP-1, leading to local chromatin relaxation. Here, we show that ATM accumulation and activity is strongly diminished at DSBs undergoing end-resection during HR. Such DSBs remain unrepaired in cells devoid of the HR factors BRCA2, XRCC3 or RAD51. Strikingly, depletion of KAP-1 or expression of phospho-mimic KAP-1 allows repair of resected DSBs in the absence of BRCA2, XRCC3 or RAD51 by an erroneous PARP-dependent alt-NHEJ process. We suggest that DSBs in heterochromatin elicit initial local heterochromatin relaxation which is reversed during HR due to the release of ATM from resection break ends. The restored heterochromatic structure facilitates HR and prevents usage of error-prone alternative processes.
Project description:Dynamic spatiotemporal modification of chromatin around DNA damage is vital for efficient DNA repair. Normal stem cells exhibit an attenuated DNA damage response (DDR), inefficient DNA repair, and high radiosensitivity. The impact of unique chromatin characteristics of stem cells in DDR regulation is not yet recognized. We demonstrate that murine embryonic stem cells (ES) display constitutively elevated acetylation of histone H3 lysine 9 (H3K9ac) and low H3K9 tri-methylation (H3K9me3). DNA damage-induced local deacetylation of H3K9 was abrogated in ES along with the subsequent H3K9me3. Depletion of H3K9ac in ES by suppression of monocytic leukemia zinc finger protein (MOZ) acetyltransferase improved ATM activation, DNA repair, diminished irradiation-induced apoptosis, and enhanced clonogenic survival. Simultaneous suppression of the H3K9 methyltransferase Suv39h1 abrogated the radioprotective effect of MOZ inhibition, suggesting that high H3K9ac promoted by MOZ in ES cells obstructs local upregulation of H3K9me3 and contributes to muted DDR and increased radiosensitivity.
Project description:Heterochromatin is a barrier to DNA repair that correlates strongly with elevated somatic mutation in cancer. CHD class II nucleosome remodeling activity (specifically CHD3.1) retained by KAP-1 increases heterochromatin compaction and impedes DNA double-strand break (DSB) repair requiring Artemis. This obstruction is alleviated by chromatin relaxation via ATM-dependent KAP-1S824 phosphorylation (pKAP-1) and CHD3.1 dispersal from heterochromatic DSBs; however, how heterochromatin compaction is actually adjusted after CHD3.1 dispersal is unknown. In this paper, we demonstrate that Artemis-dependent DSB repair in heterochromatin requires ISWI (imitation switch)-class ACF1-SNF2H nucleosome remodeling. Compacted chromatin generated by CHD3.1 after DNA replication necessitates ACF1-SNF2H-mediated relaxation for DSB repair. ACF1-SNF2H requires RNF20 to bind heterochromatic DSBs, underlies RNF20-mediated chromatin relaxation, and functions downstream of pKAP-1-mediated CHD3.1 dispersal to enable DSB repair. CHD3.1 and ACF1-SNF2H display counteractive activities but similar histone affinities (via the plant homeodomains of CHD3.1 and ACF1), which we suggest necessitates a two-step dispersal and recruitment system regulating these opposing chromatin remodeling activities during DSB repair.
Project description:The presence of H3K9me3 and heterochromatin protein 1 (HP1) are hallmarks of heterochromatin conserved in eukaryotes. The spreading and maintenance of H3K9me3 is effected by the functional interplay between the H3K9me3-specific histone methyltransferase Suv39h1 and HP1. This interplay is complex in mammals because the three HP1 isoforms, HP1?, ?, and ?, are thought to play a redundant role in Suv39h1-dependent deposition of H3K9me3 in pericentric heterochromatin (PCH). Here, we demonstrate that despite this redundancy, HP1? and, to a lesser extent, HP1? have a closer functional link to Suv39h1, compared to HP1?. HP1? and ? preferentially interact in vivo with Suv39h1, regulate its dynamics in heterochromatin, and increase Suv39h1 protein stability through an inhibition of MDM2-dependent Suv39h1-K87 polyubiquitination. The reverse is also observed, where Suv39h1 increases HP1? stability compared HP1? and ?. The interplay between Suv39h1 and HP1 isoforms appears to be relevant under genotoxic stress. Specifically, loss of HP1? and ? isoforms inhibits the upregulation of Suv39h1 and H3K9me3 that is observed under stress conditions. Reciprocally, Suv39h1 deficiency abrogates stress-dependent upregulation of HP1? and ?, and enhances HP1? levels. Our work defines a specific role for HP1 isoforms in regulating Suv39h1 function under stress via a feedback mechanism that likely regulates heterochromatin formation.
Project description:Appropriate DNA double-strand break (DSB) repair factor choice is essential for ensuring accurate repair outcome and genomic integrity. The factors that regulate this process remain poorly understood. Here, we identify two repressive chromatin components, the macrohistone variant macroH2A1 and the H3K9 methyltransferase and tumor suppressor PRDM2, which together direct the choice between the antagonistic DSB repair mediators BRCA1 and 53BP1. The macroH2A1/PRDM2 module mediates an unexpected shift from accessible to condensed chromatin that requires the ataxia telangiectasia mutated (ATM)-dependent accumulation of both proteins at DSBs in order to promote DSB-flanking H3K9 dimethylation. Remarkably, loss of macroH2A1 or PRDM2, as well as experimentally induced chromatin decondensation, impairs the retention of BRCA1, but not 53BP1, at DSBs. As a result, macroH2A1 and/or PRDM2 depletion causes epistatic defects in DSB end resection, homology-directed repair, and the resistance to poly(ADP-ribose) polymerase (PARP) inhibition-all hallmarks of BRCA1-deficient tumors. Together, these findings identify dynamic, DSB-associated chromatin reorganization as a critical modulator of BRCA1-dependent genome maintenance.
Project description:DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.
Project description:TIP60 is a lysine acetyltransferase and is known to be a haplo-insufficient tumor suppressor. TIP60 downregulation is an early event in tumorigenesis which has been observed in several cancer types including breast and colorectal cancers. However, the mechanism by which it regulates tumor progression is not well understood. In this study, we identified the role of TIP60 in the silencing of endogenous retroviral elements (ERVs). TIP60-mediated silencing of ERVs is dependent on BRD4. TIP60 and BRD4 positively regulate the expression of enzymes, SUV39H1 and SETDB1 and thereby, the global H3K9 trimethylation (H3K9me3) level. In colorectal cancer, we found that the loss of TIP60 de-represses retrotransposon elements genome-wide, which in turn activate the cellular response to pathogens, mediated by STING, culminating in an induction of Interferon Regulatory Factor 7 (IRF7) and associated inflammatory response. In summary, this study has identified a unique mechanism of ERV regulation in cancer cells mediated by TIP60 and BRD4 through regulation of histone H3 K9 trimethylation, and a new tumor suppressive role of TIP60 in vivo.