2942 Nucleic Acids Research, 2008, Vol. 36, No. 9 RESULTS PHF1 was recruited to DSB sites induced by laser micro-irradiation We established a laser micro-irradiation system that can induce diﬀerent types of DNA damage (SSBs, DSBs and base damage) in living cells and have investigated proteins responding to DNA damage (11–13). We found that proteins involved in NHEJ (Ku70, Ku80. XRCC4, Ligase4, XLF and Artemis) and HR factors (Rad52 and NBS1) are recruited to the sites by laser micro-irradiation (Supplementary Figure 1). In an eﬀort to ﬁnd new components in the cellular process responding to DSBs, we used this system to screen a group of GFP-tagged mouse PcG proteins, which are highly conserved through- out evolution and involved in transcription regulation, development, cell proliferation and tumorigenesis. We found that mPcl1, a component involved in PcG complex PRC2, was recruited to damage site immediately after irradiation with 405 nm laser in the presence of BrdU (data not shown). We observed no recruitment of other PcG proteins including mPcl2, mPcl3, PLZF, Scmh1, Ring1A, Ring1B, Mel18 and EED (data not shown). We isolated a human homolog of mPCl1, PHF1 cDNA, attached it with GFP and expressed in HeLa cell. We found that both endogenous PHF1 and GFP-tagged PHF1 accumulated at the irradiated sites and co-localized with gH2AX or with GFP-tagged Ku70 only after irradiation with 500 scans in the presence of BrdU (Figure 1A and B, see Materials and methods section). Although GFP-PHF1 accumulates at the laser irradiated site after 100 scans with BrdU pretreatment, the accumu- lation was signiﬁcantly weak (data not shown). As shown in Figure 1C, GFP-PHF1 accumulates rapidly at the irradiated sites within 1 min after irradiation and dis- sociates before 10 min. Since PHF1 dissociates very rapidly from DSBs sites, and NHEJ and HR factors remain at damage sites more than 2–4 h after irradiation (data not shown, 22), these data suggest that PHF1 is an early factor involved in DSBs damage response. PHF1 is recruited to the sites of laser micro irradiation via two independent domains of the N-terminus Tudor domain and the central region PHF1 contains two zinc ﬁnger-like PHD (derived from the name ‘plant homeodomain’) domains and a Tudor domain (Figure 2A). PHD domain is found in a number of nuclear proteins and thought to be involved in chromatin-mediated transcriptional regulation (23); the Tudor domain is found in several RNA-binding factors and believed to be chromatin-binding domain. To deter- mine which domain is responsible for the recruitment of PHF1 to laser-irradiated sites, we constructed several GFP-tagged PHF1 deletion mutants and analyzed the recruitment of these deletion mutants (Figure 2A). We found that three deletion mutants of PHF1 (aa 2–349, aa 81–431 and aa 340–567) are all recruited to irradiation sites (Figure 2B), suggesting that more than one domain are responsible for the recruitment of PHF1. More detailed analysis showed that both Tudor domain and Figure 1. Recruitment of PHF1 to the sites irradiated with 405 nm laser. HeLa cells were sensitized with BrdU and micro-irradiated. Five minutes later, the cells were photographed (for GFP-tagged protein) or ﬁxed and stained with antibody. EGFP-tagged PHF1 is recruited to laser-irradiated sites and co-localizes with gH2AX. (A) Accumulation of GFP-tagged PHF1 (EGFP-PHF1) and gH2AX. (B) Accumulation of EGFP-KU70 co-localized with endogenous PHF1. (C) Accumulation and dissociation of GFP-PHF1 at laser-irradiated sites in HeLa cells. Arrows indicate the sites of irradiation. central region (aa 340–431) are recruited to damaged sites, but PHD domain and C-terminal domain are not (Figure 2B). To further address the importance of Tudor domain and central region (aa 340–431) for the recruit- ment of PHF1 to laser-irradiated sites, we examined the recruitment of deletion mutants of PHF1 (aa 81–349 and 422–567). As expectedly, deletion mutants of PHF1 (aa 81–349 and 422–567) lacking both Tudor domain and central region (aa 340–431) showed no recruitment to damaged sites (Figure 2B). It has been reported that the two tandem Tudor domains of 53BP1 binds to methylated lysine 79 of histone H3 in response to DNA damage (24,25). Thus, our data show another example that Tudor domain plays an important role in response to DNA damage. Beside Tudor domain, the central region of PHF1 (aa 310–431) is also recruited to laser-irradiated sites, suggesting that PHF1 possesses complexity in response to DNA damage, which is not related to PHD domain. Recruitment of PHF1 is Ku70/Ku80 dependent We tested whether or not the recruitment of PHF1 to laser-irradiated sites is inﬂuenced by factors involved in NHEJ or HR. The idea is that, if PHF1 is involved in either NHEJ or HR pathway, its recruitment may be inﬂuenced by the absence of upstream factors of either pathway. PHF1 showed normal recruitment in human cell line 1022QVA (Nijmegen patient cells) mutated in NBS1, which is essential for HR (Figure 3D). PHF1 was also recruited to laser-irradiated sites in cells derived