Abstract 1999: ATR- and ATM-regulated H3K56 acetylation mediates recovery from UV-induced cell-cycle checkpoint arrest
Authors: Battu Aruna , Ray Alo , Wani Altaf A. 
Published: August 2014

DNA damage results in chromatin remodeling to allow prompt access and efficient loading of repair and checkpoint proteins at damage sites to initiate cell cycle arrest and successfully complete DNA repair. Subsequently, chromatin structure is restored and cells begin to recover from checkpoint arrest. This ‘access-repair-restore’ model of DNA repair entails a coordinated functional interaction among chromatin remodelers, histone chaperones and a variety of histone modifications. Recent studies have implicated a role for H3K56 acetylation in the DNA damage response and checkpoint recovery. In this study, we determined the role of H3K56 acetylation in response to UV-induced DNA damage. We show that H3K56 is deacetylated within 15 minutes of UV-irradiation of normal human fibroblasts (NHFs), and acetylation is promptly recovered with full restoration at 48 hours. In yeast, acetylated H3K56 is deposited at the sites of DNA damage in an ASF1-dependent manner and is involved in checkpoint recovery. Therefore, we tested whether the checkpoint kinases, ATR and ATM, regulate the acetylation of H3K56. Interestingly, H3K56 acetylation was significantly lower in Seckel cells compared to NHFs, indicating a role of ATR in regulating H3K56 acetylation even in the absence of DNA damage. On the other hand, in AT and ATM-depleted HeLa cells, the post-repair recovery of H3K56 acetylation was defective. These data indicate a contributory role of ATR and ATM in regulating H3K56 acetylation. Next, we assessed the role of histone chaperone ASF1A in deacetylation/acetylation of H3K56. shRNA-mediated knockdown of ASF1A did not show a discernable defect in the deacetylation of H3K56 in response to UV-induced DNA damage, but the restoration of H3K56 acetylation was impaired. Thus, ASF1A appears to be essential for the post-repair recovery of H3K56 acetylation. To further confirm the role of ASF1A-mediated H3K56 acetylation in checkpoint recovery, we determined the level of H2AX phosphorylation at various times following UV-irradiation in ASF1A-deficient and H3K56R mutant cells, which are defective in H3K56 acetylation. Both cell types were shown to retain phosphorylated H2AX for a longer time at damage sites as compared to the control wild-type cells, indicating the persistence of checkpoint arrest in the absence of H3K56 acetylation. Similarly, the phosphorylated state of checkpoint kinases, Chk1 and Chk2, was maintained for longer times in the absence of ASF1A and in H3K56R mutant cells. FACS analysis of cell cycle progression after UV-irradiation of both ASF1A-deficient and H3K56R mutant cells revealed a slower recovery from the checkpoint arrest. Taken together, our results demonstrate an important function of H3K56 acetylation in the recovery from DNA damage-induced cell cycle checkpoint arrest. (This work was supported by NIH grants ES2388, ES12991 and CA93413)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1999. doi:10.1158/1538-7445.AM2011-1999