Nucleolin Participates in DNA Damage Response cells were immunoprecipitated with anti-MDC1 antibody or normal rabbit IgG, and then the immuno-complexes were detected by Western blot analysis using anti-nucleolin antibody. (D) IR-induced histone release to nucleoplasm was abolished by depletion of nucleolin. U2OS cells were transfected by nucleolin siRNA. After 2 days, these cells were irradiated by 10 Gy of c-ray and were harvested at indicated times after IR. After preparation of nucleoplasm (nuclear sup) and chromatin, histone proteins in nucleoplasm were analyzed by Western blot. doi:10.1371/journal.pone.0049245.g006 Fig. 6C shows the physical interaction of nucleolin with MDC1. These results suggest that nucleolin is important for MDC1- dependent H2A/H2AX ubiquitination pathway. H2B ubiquitina- tion was also reduced in the knockdown cells (Fig. S6C), although this ubiquitination is independent of MDC1-related pathway [38]. Recently, it was reported that nucleolin is required for eviction of histone H2A/H2B complex from nucleosome through its FACT (facilitates chromatin transcription)-like activity at tran- scriptionally active sites [39]. We speculated that nucleolin is also important for histone eviction following DNA damage. Control cells showed the release of histone H2B, H3 and c-H2AX to nucleoplasm from chromatin, but nucleolin-depleted cells exhib- ited remarkably decreased these releases (Fig. 6D). However, nucleolin knockdown did not influence the expression of these histones (Fig. S6D). Taken together, nucleolin could be important for histone eviction following DNA damage and this eviction response may be important for MDC1-dependent H2A/H2AX ubiquitination pathway. Discussion We report here that nucleolin participates in the DNA double- strand break-induced DNA damage response, particularly via MDC1-dependent pathway. We indentify nucleolin as an H2AX- interacting protein and show that nucleolin can accumulate at DSB damage sites by laser micro-irradiation and ChIP analysis (Fig. 2 and S2). Although nucleolin might not be important for focus formation of early factors such as c-H2AX, NBS1/MRE11 complex and MDC1 (Fig. 3A), nucleolin could be involved in ATM-dependent phosphorylation of its substrates (Fig. 3C and S4AC). Furthermore, nucleolin-knockdown cells exhibited delayed kinetics of resolution of IR-induced c-H2AX foci indicating a deficiency in DSB repair (Fig. 5E and S5D), especially due to a defect in the HR pathway of repair (Fig. 5BC and S5A). Repression of nucleolin also decreased focus formation of 53BP1, BRCA1 and RNF168, and ubiquitination of H2A/H2AX (Fig. 3AB, 6B, S3CD and S6C). Several reports indicated that focus formation of 53BP1, BRCA1 and RNF168 are dependent on ubiquitination of H2A/H2AX in response to DNA damage [19]. RNF8 and RNF168 E3 ligases are responsible for H2A/H2AX mono- and poly-ubiquitination and these IR-induced accumula- tions at DSB damage sites require MDC1 [13–15]. MDC1 is also important for amplification of the ATM-related pathway [18], while repression of nucleolin reduced phosphorylated-ATM focus and ATM-related responses (Fig. 3, S3CD and S4AC). Moreover, MDC1 formed a complex with nucleolin dependently on DSB damage (Fig. 6C). Therefore, nucleolin could be responsible for MDC1-related DNA damage responses. This thought is in agreement with results showing that DNA damage-induced accumulation of MDC1 to chromatin was abolished in nucleo- lin-knockdown cells (Fig. S6C), although MDC1 focus formation was unchanged in their cells (Fig. 3A). Nucleolin, which is known to possess FACT-like activity through an eviction of histone H2A/ H2B complex from nucleosome at transcriptionally active sites, was also indispensable for DNA damage-induced eviction of histones (Figure 6D). Figure S2B suggests that nucleolin could recruit to DSB sites in H2AX-dependent manner. Therefore, it is suggested that histone (H2A/H2B) eviction by nucleolin, following its H2AX-dependent recruitment to DNA damage sites, could facilitate MDC1-relared DNA damage response including HR repair and ATM-mediating cell cycle checkpoint (Figure S7). Nucleolin is a major nucleolar protein containing RNA-binding motifs in the central region. Nucleolin is required for rRNA processing in nucleolus and has many other diverse functions such as transcription regulation, modulation of mRNA stability and DNA replication and recombination [25]. Recently, it was reported that nucleolin possesses FACT-like activities, stimulating SWI/SNF-mediated transfer of H2A/H2B in vitro and facilitating transcription in vitro just like FACT [39]. Hence, we hypothesize that this activity of histone transfer may be important for the DNA damage response. Several reports indicate that DSB damage stimulates histone loss around DSB damage sites in both mammalian cells and yeast using ChIP assay, and that this histone loss is followed by DSB repair response [32,40]. Indeed, Fig. 6D shows that histones were released from chromatin in response to DSB damage, but depletion of nucleolin abolished these releases. Thus, histone transfer/loss from chromatin around DNA damage sites might be important for the nucleolin-dependent regulation of DNA damage response and this nucleolin-dependent histone transfer/loss may be important for stimulation of MDC1-de- pendent DSB signaling pathway. It was also reported that nucleolin physically interacted with HR factors such as Rad51 and RPA [26,27]. Hence, these interactions may contribute to nucleolin-dependent regulation of HR independently of histone release. It has been suggested that histone modification and subsequent chromatin remodeling is closely linked with DSB repair, partic- ularly HR repair. We and other groups reported that Tip60- dependent histone acetylation is important for the HR repair pathway [7,41]. Our report showed that repression of Tip60- dependent acetylation of histone H2A/H2AX reduced focus formation of HR-related factors such as NBS1 and MRE11, and led to decreases in HR activity [7]. Murr et al. reported that a binding partner of Tip60, TRAPP-knockout mouse cells showed a decrease in histone acetylation around DSB sites, IR-induced focus formation of HR factors and HR activity [41]. Tsukuda and her colleagues reported that INO80-dependent chromatin remo- deling regulated early and late stage of mitotic homologous recombination [40]. These facts indicate the importance of histone modification and chromatin remodeling for HR repair, but their relationships with NHEJ have been unclear. Recently, Ogiwara et al. showed that CBP and p300-dependent histone acetylation could facilitate the recruitment of KU proteins and NHEJ repair activity [42]. Moreover, the chromatin remodeling factor, ACF1 was suggested to participate in NHEJ repair through initial recruitment of KU proteins to DSB sites [43]. These reports suggest that histone modification and subsequent chromatin remodeling might also contribute to the control of NHEJ repair. In fact, our data showed that nucleolin interacts with KU70 in response to DNA damage (Fig. 1C) and that nucleolin knockdown reduced both auto-phosphorylation of DNA-PKcs and NHEJ activity (Fig. S4A and 5B). Therefore, nucleolin-dependent chromatin remodeling might also be important for NHEJ pathway. As nucleolin contributes to ACF1-mediated nucleosome sliding activity [39], nucleolin may participate in NHEJ through the function of ACF1 to nucleosome. PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49245