2944 Nucleic Acids Research, 2008, Vol. 36, No. 9 Figure 4. PHF1 is associated physically with Ku70/Ku80 and may be involved in NHEJ. (A) pcDNA5/FRT vector expressing FLAG-HA tagged PHF1 was transfected into Flp-In-293 cells, and a clone stably expressing FLAG-HA-PHF1 (F-PHF1) was isolated and used for coimmunoprecipitation by anti-FLAG M2 aﬃnity gel. A clone stably expressing FLAG-HA tag alone (F-Control) was used as negative control. (B) pcDNA5/FRT vector expressing FLAG-HA tagged Ku80 was transfected into Flp-In-293 cells, and a clone stably expressing FLAG-HA-PHF1 (F-Ku80) was isolated and used for coimmunopre- cipitation by anti-FLAG M2 aﬃnity gel. (C) Real-time PCR analysis of a PHF1 stable knockdown cell line and a parallel mock knockdown cell line. HPRT was used as the control for normalization in the real-time PCR. (D) Immunoblotting analysis of PHF1 stable knockdown cell line and parallel mock knockdown cell line. Actin serves as a loading control for immunoblotting. (E) PHF1 knockdown cell showed increased sensitivity to X-Ray. Cells were plated in 6 cm dishes and irradiated with the indicated doses of ionizing irradiation. (F) Recombination frequency in HeLa cells after knockdown of PHF1. Error bars represent standard errors. PHF1 knockdown. It has been also shown that deﬁciency in NHEJ components such as Ku70, XRCC4 and DNA-PKcs results in increased levels of HR (28). Therefore, we tested whether or not downregulation of PHF1 aﬀects HR using recombination assay described previously (29). As expectedly, downregulation of PHF1 increases HR frequency several folds (Figure 4F). These data strongly suggest that PHF1 is involved in NHEJ pathway and supports its eﬃciency. PHF1 is associated with proteins responding to DNA damage besides KU and those from Polycomb group (PcG) If PHF1 is involved in damage response besides transcrip- tional regulation in human cell, PHF1 can be identiﬁed in Figure 5. Analysis of PHF1-associated proteins in HeLa cell by proteomics. (A) Putative PHF1-binding proteins identiﬁed by coimmu- noprecipitation and mass spectrometry. Among identiﬁed proteins by mass spectrometry, those related to DNA damage response and PcG are indicated. Flp-In-293 cells stably expressing FLAG-HA tagged PHF1 was used for immunoprecipitation for FLAG. Flp-In-293 cells expressing FLAG-HA-tag alone was used as negative control. (B) Candidate interacting proteins identiﬁed by nanoLC/MS/MS were further conﬁrmed using antibodies against SMC1, p53 and p46 by immunoblotting. Antibodies (IB) are indicated on the right. a proteome other than PcG group and may have other interacting proteins of damage response than KU. There- fore, a proteomics approach was undertaken to identify interacting proteins of PHF1 within human cell. A 293-cell line stably expressing PHF1 fused with FLAG-HA tags was used to immunoprecipitate the PHF1 complex by anti-FLAG antibody in the presence of ethidium bromide for exclusion of DNA-associated nonspeciﬁc proteins. Immunoprecipitated products were analyzed by SDS– PAGE, and then silver stained (Figure 5A); bands were excised, treated as described (30) and tryptic peptides were analyzed by mass spectrometry (Supplementary Table 1). It was not surprising that Suz12, EZH2 and RBBP7 (RbAp46) were identiﬁed, consistent with a previous report about Drosophila Pcl protein. More interestingly, Rad50, SMC1, DHX9 and P53, which are proteins involved in DNA damage response and repair, were also identiﬁed with high conﬁdence as coimmunoprecipitants of PHF1. The data of mass analysis were further con- ﬁrmed by western blotting with SMC1, p53 and RbAp46 antibodies (Figure 5B). Rad50 forms a complex with Mre11 and NBS1 that is essential in maintaining DNA integrity by functioning in DSBs repair, meiotic recombi- nation and telomere maintenance (31–33). DISCUSSION PcG proteins are epigenetic chromatin modiﬁers involved in gene silencing, cancer development and the mainte- nance of embryonic and adult stem cells. During screening of proteins responding to DNA damage by using laser micro-irradiation we found that mPcl1, a mouse homolog of polycomb-like protein (Pcl) of the D. melanogaster, accumulates at laser-irradiated site. It was previously shown that the Drosophila Pcl protein is associated with