or a binding partner, YY1, increased cellular sensitivity toward DNA-damaging agents, and that both INO80 and YY1 are essential for HR repair [11]. Downregulation of Brg1, a compo- nent of human SWI/SNF1 complex, results in inefficient DSB repair and increased DNA damage sensitivity [12]. Thus, these and additional reports suggest that both histone modification and chromatin remodeling are important for the maintenance of genomic stability in the face of both endogenous and exogenous DNA damage. Recently, histone ubiquitination has emerged as an important event in the DNA damage response (DDR). Histone H2A and its variant H2AX are both ubiquitinated by RNF8 and RNF168 E3 ligase, in a MDC1 (mediator of DNA damage checkpoint 1)- dependent manner, following generation of DSBs [13–15]. This ubiquitinated H2A/H2AX recruits HR-related factors such as RAP80, Abraxas and BRCA1, leading to activation of HR machinery [16]. MDC1 was identified as a binding partner for the hMRE11/hRAD50/NBS1 (MRN) complex and contains a fork- head-associated (FHA) domain and two BRCA1 carboxy-terminal (BRCT) domains [17]. The binding of MDC1 to c-H2AX through its tandem BRCT domains is important for its recruitment and accumulation at DSB sites, which, in turn, facilitates activation of ATM-dependent cell cycle checkpoint and HR repair [18]. Furthermore, MDC1 is phosphorylated by ATM in response to DSB damage and this phosphorylation is responsible for the recruitment and accumulation of RNF8 and RNF168 to DSB sites [19]. These facts indicate that MDC1 is an important regulatory factor for histone H2A ubiquitination in the DNA damage response and might function in chromatin remodeling in response to DNA damage through histone ubiquitination. H2AX is one of the variants of histone H2A and comprises 2– 25% of the H2A complement in mammalian chromatin. As previously reported by us, H2AX is rapidly phosphorylated at Serine139 in an ATM-dependent manner at the sites of DSBs [20] and phosphorylated H2AX (c-H2AX) interacts with NBS1, MDC1 and BRCA1, thereby promoting their accumulation at DSB sites [21,22]. Hence, H2AX-deficient cells are defective in the formation of DSB-induced nuclear foci of several DNA damage-related (DDR) proteins such as NBS1 [22–24]. We also reported that this phosphorylation of H2AX contributes to the activation of ATM kinase [24]. Besides phosphorylation, histone H2AX is subject to other modifications such as acetylation, methylation, and ubiquitination. As mentioned above, acetylation and ubiquitination of H2A/H2AX are important for accumula- tion of DDR proteins and subsequent initiation of HR repair. Furthermore, c-H2AX is also important for the recruitment of chromatin remodeling factors such as yeast Ino80 to DSB sites by direct binding through its phosphorylation site [9]. These facts suggest that H2AX is one of the key regulators of chromatin remodeling via its phosphorylation on serine139. In order to define the H2AX-dependent chromatin remodeling pathway, we attempted to identify components of the c-H2AX complex which forms at DSB sites using GST-pull-down assays coupled with proteomics analysis. As a result, we identified nucleolin as a candidate of H2AX-interacting protein and investigated its role in DDR. Nucleolin accumulated into DSB damage sites in H2AX- dependent manner. Depletion of nucleolin by siRNA reduced accumulation of several DDR proteins and resulted in reduction of ATM-dependent cell cycle checkpoint and DSB repair. Nucleolin also seemed to be responsible for MDC1-dependent histone ubiquitination and other MDC1-related responses. Thus, nucleo- lin could be important for H2AX/MDC1-related DNA damage responses, and we discuss this novel function of nucleolin in DSB damage response and chromatin remodeling. Nucleolin Participates in DNA Damage Response Results Identification of Nucleolin as an H2AX-associated Protein In order to identify proteins that bind to H2AX, especially phosphorylated H2AX (c-H2AX), we designed GST-fused H2AX and GST-fused phospho-mimic H2AX, whose serine at position 139 is substituted to glutamic acid. These recombinant proteins were used to ‘‘pull down’’ possible associating proteins from nuclear extracts of irradiated HeLa cells. Candidate H2AX- interacting proteins were identified by peptide Mass fingerprint analysis (Fig. S1A). One of the identified proteins was the approximately 100 kDa nucleolar protein, nucleolin (Fig. 1A and S1B). Nucleolin has RNA binding activity and is important for protein translation processes [25]. Recently, it was reported that nucleolin physically interacts with Rad51 and Replication Protein A (RPA) and contributes to the tumor repressor function of p53 [26–28], suggesting that nucleolin may be an important player in the DSB damage response. We first confirmed whether nucleolin is actually part of the GST-H2AX-pulldown complex (Fig. 1B). Phospho-mimic H2AX (S139E) precipitated nucleolin from nuclear extracts, but unexpectedly, wild type H2AX also pulled down nucleolin. Remarkably NBS1 was also co-precipitated by phospho-mimic H2AX from nuclear extract of irradiated cells. As nucleolin has been reported to interact with histone H2A [25], we also tried GST-H2A-pulldown assay (Fig. S1DE). We noticed that the band patterns of precipitated proteins were different between GST-H2A and GST-H2A-S139E around 100 kDa (* in Fig. S1D), but Western blot analysis showed that GST-H2A precipitated similar amount of nucleolin to GST-H2AX (Fig. S1E). We next examined the endogenous interaction between nucleolin and c- H2AX. Anti-nucleolin antibody co-precipitated c-H2AX and the NHEJ repair factor, KU70 from irradiated cells (Fig. 1C), while another major ribosomal protein S6 did not interact with neither c-H2AX nor Ku70 (Fig. S1C). Anti-c-H2AX antibody also preferably precipitated nucleolin from irradiated cell extracts (Fig. 1D). As KU70/KU80 complex are rapidly recruited to DSB ends, this result indicates that nucleolin might interact with c- H2AX at DSB sites. Nucleolin Accumulates at DNA Damage Sites in an H2AX- dependent Manner As a large number of DDR proteins recruited to DNA damage sites can be visualized as nuclear ‘‘foci’’, we investigated the focus formation of nucleolin following generation of DNA damage (Fig. 2A). As nucleolin is a nucleolar protein, almost all GFP- nucleolin is confined to the nucleoli in the absence of DNA damage. DNA damage induction by camptothecin (CPT) and etoposide did not induce nuclear focus formation of nucleolin, but triggered its relocation from nucleoli to the nucleoplasm. We also examined the re-localization of endogenous nucleolin by immu- nofluorescence using anti-nucleolin antibody, but failed to observe focus formation in response to c-rays, CPT, or etoposide (data not shown). Although several DDR proteins, such as the NHEJ protein Ku70/80, are recruited to DSB sites, their recruitment cannot be visualized as nuclear foci, perhaps because they are localized closer to the DSB and are not spread out over megabases of chromatin as with c-H2AX [29]. In these cases, their recruitment to DSB sites could be visualized using the laser micro-irradiation method [30,31]. Therefore, we investigated whether nucleolin is recruited to DNA damage sites induced by laser micro-irradiation. Upon irradiation of nucleoplasm with 405 nm of laser, GFP-nucleolin relocated rapidly to the irradiated area (Fig. 2B). However, micro- irradiation of the nucleolus did not trigger relocation to irradiated PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49245