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.histone methylation | homologous recombination D NA double-strand breaks (DSBs) are toxic and must be repaired to maintain genomic stability. Detection of DSBs requires recruitment of the mre11-rad50-nbs1 (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 ATM's kinase activity (4-6) 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 (γH2AX) extending for hundreds of kilobases along the chromatin (7,8). Mdc1 then binds to γH2AX, 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 (12-15), leading to the formation of open, flexible chromatin domains adjacent to the break (12, 13). These open chromatin structures then facilitate histone ub...

Chromatin remodeling during DNA double-strand break (DSB) repair is required to facilitate access to and repair of DSBs. This remodeling requires increased acetylation of histones and a shift in nucleosome organization to create open, relaxed chromatin domains. However, the underlying mechanism driving changes in nucleosome structure at DSBs is poorly defined. Here, we demonstrate that histone H2A.Z is exchanged onto nucleosomes at DSBs by the p400 remodeling ATPase. H2A.Z exchange at DSBs shifts the chromatin to an open conformation, and is required for acetylation and ubiquitination of histones and for loading of the brca1 complex. H2A.Z exchange also restricts single-stranded DNA production by nucleases and is required for loading of the Ku70/80 DSB repair protein. H2A.Z exchange therefore promotes specific patterns of histone modification and reorganization of the chromatin architecture, leading to the assembly of a chromatin template which is an efficient substrate for the DSB repair machinery.

Senescence induction could be used as an effective treatment for hepatocellular carcinoma (HCC). However, major senescence inducers (p53 and p16 Ink4a) are frequently inactivated in these cancers. We tested whether transforming growth factor-b (TGF-b) could serve as a potential senescence inducer in HCC. First, we screened for HCC cell lines with intact TGF-b signaling that leads to small mothers against decapentaplegic (Smad)-targeted gene activation. Five cell lines met this condition, and all of them displayed a strong senescence response to TGF-b1 (1-5 ng/mL) treatment. Upon treatment, c-myc was down-regulated, p21Cip1 and p15 Ink4bwere up-regulated, and cells were arrested at G 1 . The expression of p16 Ink4a was not induced, and the senescence response was independent of p53 status. A short exposure of less than 1 minute was sufficient for a robust senescence response. Forced expression of p21Cip1 and p15Ink4b recapitulated TGF-b1 effects. Senescence response was associated with reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) induction and intracellular reactive oxygen species (ROS) accumulation. The treatment of cells with the ROS scavenger N-acetyl-L-cysteine, or silencing of the NOX4 gene, rescued p21 Cip1 and p15 Ink4b accumulation as well as the growth arrest in response to TGF-b. Human HCC tumors raised in immunodeficient mice also displayed TGF-b1-induced senescence. More importantly, peritumoral injection of TGFb1 (2 ng) at 4-day intervals reduced tumor growth by more than 75%. In contrast, the deletion of TGF-b receptor 2 abolished in vitro senescence response and greatly accelerated in vivo tumor growth. Conclusion: TGF-b induces p53-independent and p16 Ink4a -independent, but Nox4-dependent, p21 Cip1-dependent, p15 Ink4b-dependent, and ROS-dependent senescence arrest in well-differentiated HCC cells. Moreover, TGF-b-induced senescence in vivo is associated with a strong antitumor response against HCC. (HEPATOLOGY 2010;52:966-974)