a 1 MDa complex containing the members of PcG, ESC, E(Z), SU(Z)12, p55 and RPD3, and, furthermore. Pcl is present in another unknown complex, which does not contain any component of the 600 kDa ESC/E(Z) complex (34). Therefore, we were interested in the mechanisms and function of the damage response and isolated a human homolog of Pcl1, PHF1. PHF1 was identiﬁed several years ago as a human homolog of Drosophila Pcl that locates on 6p21.3, belonging to MHC classII region (35). Although PHF1 shares signiﬁcant sequence similarity to the Drosophila Pcl, and was shown recently to be involved in tumorigenesis and/or tumor progression of endometrial stromal sarcoma by genetic analysis (36), its function was still unknown. In this study, we identiﬁed PHF1 as a new component in DSBs damage response, possibly involved in the repair pathway of DSBs. Endogenous and GFP-tagged PHF1 were recruited immediately to the sites irradiated with laser micro-irradiation in HeLa cells (Figure 1). Since PHF1 accumulates at the site irradiated with higher laser dose in the presence of BrdU and PHF1 neither accumulates at the site irradiated with lower laser dose of the laser light, nor responds to UV-irradiation (data not shown), PHF1 is possibly involved in DSBs damage response. Although the response of endogenous PHF1 was only weakly shown by antibody (Figure 1), it is probably due to low sensitivity of the antibody and/or rapid dissociation of PHF1 from the accumulated sites as shown by the results obtained with GFP-tagged protein (Figure 1C). PHF1 did not accumulate at irradiated sites in Ku-deﬁcient XR-V15B cells, but it accumulated in V15B cells expressing Ku80 (Figure 3B). These data suggest that PHF1 is recruited under the inﬂuence of Ku proteins to DSBs. In contrast to Ku-deﬁcient cells, PHF1 accumulation was not aﬀected in other mutant cells, which are defective in DNA-PKcs, XRCC4 and NBS1 (Figure 3C–E). We thought, therefore, that there might be some physical and/or functional interactions between PHF1 and Ku70/Ku80. Co-IP experiments showed that PHF1 is indeed physically associated with Ku70/Ku80 (Figure 4A and B). In accor- dance with these data, siRNA-mediated downregulation of PHF1 provided the host HeLa cells with a mild but consistent cell sensitivity to X-ray (Figure 4E). Further- more, the downregulation of PHF1 leads the cells to an enhanced HR frequency (Figure 4F), which provides further evidence that PHF1 is involved in DSBs repair and supports NHEJ. Like other NHEJ factors PHF1 is recruited to DSBs sites both G1 and S/G2 phases (Figure 3E). PHF1 contains two zinc ﬁnger-like PHD domains and a Tudor domain (Figure 2A). Tudor domain of 53BP1 interacts with methylated histones and mediates its recruitment to DSBs sites (24). Recently, PHD domain was shown to promote both gene expression and repres- sion through an interaction with H3K4me3 tails (37–40). The data reported here showed that PHD domain is not responsible for the recruitment of PHF1 to DSBs sites, while Tudor domain and central region of PHF1 (310–431) are responsible for the recruitment (Figure 2A). Preliminary data suggest that neither of the domains interacts with Ku80 (data not shown) but the whole Nucleic Acids Research, 2008, Vol. 36, No. 9 2945 PHF1 interacts with Ku proteins, suggesting that accu- mulation of PHF1 at DSBs requires Ku protein and the two domains have individual binding activities to DSBs. Further analysis is necessary to understand the mecha- nisms behind the recruitment of PHF1 to DSBs. Besides Ku70/Ku80, PHF1 is also associated with vari- ous proteins involved in the response to DSBs and other genomic maintenance mechanisms, Rad50, DHX9, SMC1 and p53 (Figure 5). Rad50 forms a complex with Mre11 and NBS1 that is essential in maintaining DNA integrity by functioning in DSBs repair, meiotic recombination and telomere maintenance (31–33). SMC1 and SMC3 consti- tute the core of the cohesion complexes which play an important role in the repair of DNA DSBs from yeast to human, and SMC1 and SMC3 also interact with Mre11- Rad50 (41–44). DHX9 is a DNA- and RNA-dependent helicase associated directly with g-H2AX (45). As a tumor suppressor, p53 plays a central role in the DNA damage response involved in multiple signaling pathways (46,47). Rad50, SMC1 and p53 were also demonstrated to be recruited to DSBs sites induced by laser micro-irradiation (44,48). The SMC1/SMC3 cohesion complex facilitates DSBs repair by HR and holds sister chromatids together locally at DSBs to allow strand invasion and exchange with the sister chromatid template (41–43). Defect in Rad50 inﬂuences phosphorylation of SMC1 and reduces HR (31,49). Moreover, another binding protein of PHF1, RbAp46 is a component of histone deacetylase complexes and is involved in chromatin remodeling, interacts with BRCA1 (50). These data suggest that PHF1 may play a role in HR as well. Further analysis is required to understand possible functions of PHF1 in the damage response. While we do not know the exact function of PHF1 in the DSBs repair yet, we demonstrated for the ﬁrst time in mammalian cells that PHF1, as known as a PcG protein, is involved in DSBs response and possibly in its repair. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank Drs Maria Jasin and Hiroshi Tauchi for providing us with expression vector pCMV3nls-I-SceI and HeLa cell line containing a stably integrated copy of recombination reporter vector SCneo. This work was supported in part by a grant from the Genome Network Project, a Grant-in-Aid for Scientiﬁc Research (A), 16201010, and Priority Areas Cancer, 18012003 from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to A.Y.). Funding to pay the Open Access publication charges for this article was provided by the Genome Network Projects. Conﬂict of interest statement. None declared. REFERENCES 1. Sonoda,E., Hochegger,H., Saberi,A., Taniguchi,Y. and Takeda,S. (2006) Diﬀerential usage of non-homologous