2940 Nucleic Acids Research, 2008, Vol. 36, No. 9 With the aim of identifying new factors involved in DSBs damage response and repair of mammalian cells, we screened a number of proteins involved in chromatin remodeling and regulation by using laser micro- irradiation system (11–13). PcG proteins are epigenetic chromatin modiﬁers involved in transcription regulation, maintenance of embryonic and adult stem cells and cancer development (14,15). PcG genes were ﬁrst identiﬁed by their requirement for the maintenance of the stable repression of Hox genes during the development of Drosophila melanogaster and are highly conserved throughout evolution. In mammals, PcG genes are also implicated in Homeobox (Hox) gene regulation. Their biological activity lies in stable silencing of speciﬁc sets of genes through chromatin modiﬁcations. Recently, emerg- ing evidence implicates the PcG proteins in cellular proliferation and tumorigenesis (15–18). Furthermore, overexpression of a PcG protein, EZH2, in breast epithelial cells reduced Rad51 paralogs both in the mRNA and protein levels which are required for proper HR DNA repair (19), and heterozygosity for mutations in either extra sex combs (Esc) or Enhance of Polycomb [E(PC)] increases the lever of HR and enhances genome stability in somatic cells of D. melanogaster (20). Laser micro-irradiation makes it possible to introduce various types of DNA damage at restricted regions in the nucleus of a single cell and to analyze the response of proteins to the damage with antibody by immuno-staining or with transfected GFP-tagged proteins under micro- scope in a real-time image. So far as laser light dose and exposed time are extremely limited, there is no eﬀect of heat production. The major product of irradiation with UVA laser light is DNA damage, because UVA laser light is either absorbed directly by DNA or by photosensitizers around DNA creating radicals, which attack DNA. Therefore, major types of DNA damage produced by UVA laser irradiation are oxidative ones, and with increasing laser dose and by addition of photosensitizers, DNA single-strand breaks, DSBs and base damage can be produced eﬀectively (11,12,21). For the reason that both PcG proteins play important roles in tumorigenesis, and some of PcG proteins are shown by genetic analysis to be involved in DSBs damage response, we analyzed whether or not PcG proteins are directly involved in the response to DSBs. We found that PHD ﬁnger Protein 1 (PHF1) is recruited rapidly to DSBs sites, that is dependent on Ku70/Ku80, and demonstrated that PHF1 is associated with Ku70/Ku80, Rad50, DHX9, SMC1 and P53 besides PcG proteins. Furthermore, knockdown of PHF1 leads to X-ray sensitivity and increases HR frequency. These data suggest that PHF1 is involved in DSBs damage response and may play an important role in NHEJ and maintaining genome stability. MATERIALS AND METHODS Construction of plasmids for expression of various genes Plasmids expressing human genes encoding KU70, KU80, XRCC4, LigaseIV, Artemis, XLF, RAD52 and NBS1 were constructed by cloning cDNA ampliﬁed from HeLa cells. PHF1 (isoform 2) was ampliﬁed from a human cDNA clone (no. MGC: BC008834). We modiﬁed the multiple cloning sites of vectors EGFP-C1, EGFP-N1 to introduce various cDNAs attached with an in-frame XhoI or SalI site at the start and NotI site at the stop codons. Deletion fragments of PHF1 were generated by PCR ampliﬁcation, and then cloned into vectors. All constructs were veriﬁed by sequencing. Cell lines, culture and transfection The following cell lines were used in this study: HeLa; V79B; XR-V15B (Ku80 / CHO cell line); CHO9; XR-C1 (DNA-PKcs / CHO cell line); XR-1 (XRCC4 / CHO cell line); 1022QVAP81 (NBS1-deﬁcient human cell line); Flp-In-293 (Invitrogen), All cell lines were propagated in Dulbecco’s modiﬁed-MEM (Nissui), supplemented with 1 mM L-glutamine and 10% fetal bovine serum at 378C and 5% CO2. For suspension culture, cells were grown in Joklik medium (Sigma) supplemented with 5% fetal bovine serum at 378C in spinner ﬂask. Caps of spinner ﬂask should be tightly closed and density of cells was kept between 2 and 6 105/ml. For UVA-laser irradiation, cells were plated in glass-bottomed dishes (Matsunami Glass) and transfected with expression vectors using fuGene6 (Roche), according to the manufacturer’s protocol. Microscopy and UVA-laser irradiation Fluorescence images were obtained and processed using a confocal scanning laser microscopy system (FV-500, Olympus). UVA-laser irradiation was used to induce DSBs in cultured cells as described previously (11–13). Brieﬂy, cells in glass-bottomed dishes were micro-irradiated with a 405 nm pulse laser (Olympus) along a user-deﬁned path. Laser was focused through a 40 objective lens and the treatment dose was controlled by number of scans used. A single laser scan at full power delivers about 1600 nW. Cells were treated with 10 nM 5-bromo-2-deoxyuridine (BrdU) during 24 h prior to irradiation. Given the low treatment doses and the wavelength used, the inﬂuence of the laser light on DNA is mainly indirect via photo- sensitization of natural or added (BrdU) chromophores near the DNA within cell. The irradiation dose was ﬁxed in the experiments as 500 scans in the presence of BrdU, which produces more than 103 DSBs per cell, which cor- responds to 30 Gy of X-ray irradiation to a human cell. The number of DSBs was determined by comparison of the number of gH2AX produced by laser micro-irradiation with that produced by X-Ray irradiation, based on the method reported before (48). Immunofluorescence Cells were ﬁxed in cold methanol/acetone (1:1) for 10 min at 208C and probed with mouse anti-PHF1 (1:30; M01, Abnova), mouse anti-gH2AX (1:400; jbw103, Upstate). The secondary antibody used was Alexa ﬂuor 594 anti-mouse IgG (1:400; molecular Probes). Nuclear DNA was stained with 40,60-diamidino-2-phenylindole (DAPI; 0.5 mg/ml, Wako). Fluorescence microscopy was