Oncogene (2011) 30, 2135–2146 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Histone acetylation by CBP and p300 at double-strand break sites facilitates SWI/SNF chromatin remodeling and the recruitment of non-homologous end joining factors H Ogiwara1,AUi1,2,3, A Otsuka1,2, H Satoh4, I Yokomi4,5, S Nakajima3, A Yasui3, J Yokota2 and T Kohno1 1Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan; 2Division of Multistep Carcinogenesis, National Cancer Center Research Institute, Tokyo, Japan; 3Department of Molecular Genetics, Institute of Development, Division of Dynamic Proteome, Aging and Cancer, Tohoku University, Sendai, Japan; 4Laboratory of Tumor Cell Biology, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan and 5Department of Pharmacology, School of Medicine, St Marianna University, Kanagawa, Japan Non-homologous end joining (NHEJ) is a major repair Introduction pathway for DNA double-strand breaks (DSBs) generated by ionizing radiation (IR) and anti-cancer drugs. There- Non-homologous end joining (NHEJ) is a repair path- fore, inhibiting the activity of proteins involved in this way for DNA double-strand breaks (DSBs) that joins pathway is a promising way of sensitizing cancer cells two broken DNA ends without the need for sequence to both radiotherapy and chemotherapy. In this study, we homology. DSBs caused by ionizing radiation (IR) are developed an assay for evaluating NHEJ activity against preferentially repaired through NHEJ (Burma et al., DSBs in chromosomal DNA in human cells to identify the 2006; Lieber, 2008). Therefore, suppression of NHEJ chromatin modification/remodeling proteins involved in is a promising therapy that may sensitize cancer cells to NHEJ. We showed that ablating the activity of the homo- IR (Helleday et al., 2008). In addition, DSBs caused by logous histone acetyltransferases, CBP and p300, using anticancer drugs such as the topoisomerase II inhibitor, inhibitors or small interfering RNAs-suppressed NHEJ. etoposide, may also be repaired through NHEJ (Adachi Ablation of CBP or p300 impaired IR-induced DSB repair et al., 2003); therefore, suppression of NHEJ is also and sensitized lung cancer cells to IR and the anti-cancer a promising approach to sensitizing cancer cells to these drug, etoposide, which induces DSBs that are repaired by drugs (Zhao et al., 2006; Helleday et al., 2008). In vitro NHEJ. The CBP/p300 proteins were recruited to sites of studies have elucidated the molecular processes under- DSBs and their ablation suppressed acetylation of lysine 18 lying NHEJ of DNA ends, in which KU70/80 and within histone H3, and lysines 5, 8, 12, and 16 within DNA-dependent protein kinases (DNA-PKcs) function histone H4, at the DSB sites. This then suppressed the as core proteins (Burma et al., 2006; Lieber, 2008). recruitment of KU70 and KU80, both key proteins for Chemicals that inhibit DNA-PKcs activity and sensitize NHEJ, to the DSB sites. Ablation of CBP/p300 also cancer cells to IR and/or etoposide are awaiting eval- impaired the recruitment of BRM, a catalytic subunit of uation for clinical application (Helleday et al., 2008). the SWI/SNF complex involved in chromatin remodeling Thus, identification of novel proteins involved in NHEJ at DSB sites. These results indicate that CBP and p300 will help to develop methods for sensitizing cancer cells function as histone H3 and H4 acetyltransferases at DSB to both radiotherapy and chemotherapy. sites in NHEJ and facilitate chromatin relaxation. There- Chromosomal DNA and histones form a highly fore, inhibition CBP and p300 activity may sensitize cancer condensed structure known as chromatin. The accessi- cells to radiotherapy and chemotherapy. bility of proteins to chromosomal DNA in vivo is Oncogene (2011) 30, 2135–2146; doi:10.1038/onc.2010.592; required for a variety of intracellular processes, includ- published online 10 January 2011 ing DSB repair, and is regulated by two general chromatin remodeling mechanisms, histone modifica- Keywords: non-homologous end joining; DNA double- tion and alteration of the nucleosomal position (Osley strand break; DNA repair; chromatin; histone acetyl- and Shen, 2006). Acetylation of histones located at transferase DSB sites by histone acetyltransferases (HATs) is a critical chromatin modification required for DSB repair. In particular, the N-terminal lysine residues of histones H3 and H4 are acetylated during DSBs (Bird et al., Correspondence: Dr T Kohno, Division of Genome Biology, National 2002; Tamburini and Tyler, 2005). Consistent with this, Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo NuA4-Tip60 HAT acetylates histone H4 at DSB sites 104-0045, Japan. E-mail: [email protected] and facilitates DSB repair (Bird et al., 2002; Murr et al., Received 15 July 2010; revised 1 December 2010; accepted 3 December 2006). In addition to HATs, the SWI2/SNF2 super- 2010; published online 10 January 2011 family (INO80, SWR1, SWI/SNF and RSC) chromatin Role of CBP/p300 in non-homologous end joining H Ogiwara et al 2136 remodeling complexes are recruited to DSB sites and production of a transcript that enables translation of facilitate alterations in the nucleosomal position. enhanced green fluorescent protein (EGFP) instead Furthermore, the INO80, SWR1 and RSC complexes of the herpes simplex virus-thymidine kinase protein. are required for the recruitment of KU70/80 to DSB Therefore, the efficiency of NHEJ can be assessed sites (Shim et al., 2005; van Attikum et al., 2007). It is by monitoring EGFP production. In addition, the also suggested that the SWI/SNF complex is recruited DSBs produced by I-SceI and NHEJ of the two broken to DSB sites, where it interacts with acetylated DNA strands can be monitored by quantitative PCR histone proteins and g-H2AX, a phosphorylated (As shown in Figure 1a, ‘the proportion of uncut DNA’ H2AX protein generated upon DSBs (Lee et al., 2010). is used to assess the efficiency of DSB generation by Taken together, these studies suggest that histone I-SceI and ‘the proportion of joined DNA’ is used to acetylation facilitates chromatin remodeling and that assess the ligation efficiency of the DNA ends). the resulting ‘relaxed’ chromatin enables the recruitment We isolated two individual H1299 human lung cancer of DNA repair proteins to DSB sites. However, the cell clones, H1299dA3-1 #1 and #2, that stably carried HATs and chromatin remodeling complexes involved the IRES-TK-EGFP DNA within their genome in NHEJ have not been fully understood. In addition, (Supplementary Figure S1A and S1B). Fluorescence- the effect of inhibiting these proteins on the radio- activated cell sorting analysis showed that the propor- sensitivity and chemosensitivity of cancer cells remains tion of cells expressing EGFP increased 24–72 h after largely unknown. transfection of the I-SceI expression plasmid in both In this study, we developed an assay system to clones (Figure 1b and Supplementary Figure S1C). evaluate NHEJ activity after DSBs in chromosomal Quantitative PCR analysis revealed that the proportion DNA in human cells. Using this system, we found that of joined DNA increased 12–48 h post transfection, two HAT inhibitors, anacardic acid and curcumin, both whereas that of uncut DNA decreased during the 24 h known to radio-sensitize cancer cells (Khafif et al., 2005; post transfection (Figures 1c and d). Therefore, DSBs Sun et al., 2006; Javvadi et al., 2008), suppress NHEJ were introduced by I-SceI, and the subsequent joining of DSBs. The CBP and p300 proteins are homologous of the two broken DNA ends occurred in both the HATs, known to function as transcription co-activators, H1299dA3-1 #1 and #2 clones in vivo. Nucleotide and their enzymatic activity is inhibited by anacardic sequencing of the joined DNA revealed that the ligation acid and curcumin (Karamouzis et al., 2007). We required no (or very little) sequence homology between showed that CBP and p300 function as histone H3 the DNA ends (Supplementary Figure S1D), indicating and H4 acetyltransferases at DSB sites during NHEJ, that the DNA ends were joined via NHEJ. and facilitate the recruitment of KU70/80 proteins in We next examined the effect of inhibiting DNA-PKcs cooperation with the SWI/SNF chromatin remodeling activity on the joining of DNA ends using this assay complex. Ablation of CBP and p300 caused radio- system. In both the H1299dA3-1 #1 and #2 clones, the sensitization of lung cancer cells and sensitized the cells proportion of both EGFP-positive cells and joined to etoposide. These results indicate that the CBP and DNA decreased after treatment with the DNA-PKcs p300 proteins are involved in NHEJ in vivo and act as inhibitor, NU7026, at a concentration wherein phos- histone H3 and H4 acetyltransferases, facilitating phorylation of the serine 2056 residue of the DNA-PKcs chromatin relaxation. Inhibition of CBP and p300 protein (activated by autophosphorylation) by IR is activity may, therefore, be a way to sensitize cancer suppressed. This occurred without any increase in the cells to radiotherapy and chemotherapy. proportion of uncut DNA (Figures 1e–g and Supple- mentary Figure S1E). The proportion of EGFP-positive cells was also decreased by small interfering RNA Results (si)RNA-mediated ablation of DNA-PKcs (Figure 1h). These results confirmed that the two DNA ends Development of an NHEJ assay system for DSBs produced by I-SceI digestion were joined through in chromosomal DNA NHEJ, and indicated that a reduction in NHEJ activity The assay system for evaluating NHEJ activity against by treatment with inhibitors and siRNAs directed DSBs in chromosomal DNA in living human cells is against other proteins involved in NHEJ should also presented in Figure 1a. The IRES-TK-EGFP plasmid, be detectable by this assay. which contains two recognition sites for I-SceI endo- nuclease (Jasin, 1996) in the reverse direction, was integrated into the chromosomal DNA of human cells Suppression of NHEJ in vivo by ablation of CBP or p300 as a substrate for DSBs and subsequent NHEJ repair.