Oncogene (2003) 22, 8931–8938 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc A member of the Pyrin family, IFI16, is a novel BRCA1-associated protein involved in the p53-mediated apoptosis pathway Jason A Aglipay1, Sam W Lee2, Shinya Okada1, Nobuko Fujiuchi1, Takao Ohtsuka2, Jennifer C Kwak2, Yi Wang3,4, Ricky W Johnstone5, Chuxia Deng6, Jun Qin3,4 and Toru Ouchi*,1 1The Derald H. Ruttenberg Cancer Center, The Mount Sinai School of Medicine, New York University, New York, NY, USA; 2Cancer Biology Program, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA; 3Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA; 4Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA; 5Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Victoria, Australia; 6Genetics of Development and Disease Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA We identified IFI16 as a BRCA1-associated protein C-terminal BRCT domains (Miki et al., 1994; Koonin involved in p53-mediated apoptosis. IFI16 contains the et al., 1996; Bork et al., 1997). Pyrin/PAAD/DAPIN domain, commonly found in cell Recently, mass-spectrometric analysis ofBRCA1- death-associated proteins. BRCA1 (aa 502–802) inter- interacting proteins revealed the presence of BRCA1- acted with the IFI16 Pyrin domain (aa 1–130). We found associated genome surveillance complex (BASC) (Wang that IFI16 was localized in the nucleoplasm and nucleoli. et al., 2000). This large complex includes ATM/ATR Clear nucleolar IFI16 localization was not observed in kinases, RAD51, MSH2, MSH6, MLH1, BLM and the HCC1937 BRCA1 mutant cells, but reintroduction of RAD50/Mre11/NBS complex, which are all involved in wild-type BRCA1 restored IFI16 nuclear relocalization homologous or nonhomologous recombination; coloca- following IR (ionizing radiation). Coexpression of IFI16 lization ofBRCA1 to these proteins has been demon- and BRCA1 enhanced DNA damage-induced apoptosis in strated by immunocytochemical analysis. A possible mouse embryonic fibroblasts from BRCA1 mutant mice role ofBRCA1 in the DNA repair machinery has also expressing wild-type p53, although mutant IFI16 deficient been shown by reintroduction ofwild-type or mutant in binding to BRCA1 did not induce apoptosis. Further- alleles ofBRCA1 into the HCC1937 breast cancer cell more, tetracycline-induced IFI16 collaborated in inducing line, which expresses truncated BRCA1 protein (Scully apoptosis when adenovirus p53 was expressed in DNA- et al., 1999). Thus, cells into which wild-type BRCA1 damaged p53-deficient EJ cells. These results indicate a has been reintroduced recovered resistance to DNA BRCA1-IFI16 role in p53-mediated transmission of DNA damage-induced cell death. Significantly, reintroduction damage signals and apoptosis. ofany patient-derived BRCA1 mutants examined has Oncogene (2003) 22, 8931–8938. doi:10.1038/sj.onc.1207057 failed to confer resistance to DNA damage on the recipient cells to the same degree as reintroduction of Keywords: BRCA1; IFI16; p53; apoptosis wild-type BRCA1, suggesting that the integrity provided by the complete structure ofthe BRCA1 protein is necessary for this activity. A significant body ofevidence also indicates that Introduction BRCA1 regulates cellular transcription factors such as p53, STAT1 and ZBRK1 (Ouchi et al., 1998; Zhang Germline mutations in the BRCA1 locus predispose et al., 1998; Ouchi et al., 2000; Zheng et al., 2000). women to early-onset, hereditary breast cancer (Miki Functional interaction between BRCA1 and p53 has et al., 1994). Mutations in BRCA1 have been found in been shown by murine experiments, where loss ofp53 approximately 90% offamilial breast and ovarian may lead to BRCA1-mutated breast cancer development cancers (reviewed by Alberg and Helzlsouer, 1997; and regulation ofapoptosis (Xu et al., 1999, 2001). A Nathanson et al., 2001). BRCA1 encodes a large, role ofBRCA1 in gene expression has been further multifunctional nuclear phosphoprotein of 1863 amino demonstrated by cDNA microarray analysis, in which acids containing the N-terminal RING finger and the tetracycline-regulatable BRCA1-induced GADD45 and expression ofseveral cellular genes (Harkin et al., 1999). IFI16 has been identified as a target ofinterferon (IFN) a and g and is a member ofthe HIN-200 family *Correspondence: T Ouchi, The Derald H Ruttenberg Cancer Center, (reviewed in Johnstone and Trapani, 1999). The Mount Sinai School ofMedicine, New York University, Box 1130, One Gustave L Levy Place, New York, NY 10029, USA; GAL4DBD-fused full-length IFI16 acts as a potent E-mail: [email protected] transcription repressor when positioned in proximity to Received 9 April 2003; revised 23 July 2003; accepted 31 July 2003 a promoter containing consensus GAL4DBD binding IFI16 is a novel BASC inducing p53-mediated cell death JA Aglipay et al 8932 sequences (Johnstone et al., 1998); however, it is unclear containing nuclear protein IFI16 (Trapani et al., 1992; whether IFI16 is involved in chromatin remodeling. Aravind et al., 2001), which coimmunoprecipitated with Each ofthe 200 amino acid repeat regions contains such BRCA1 when exposed to anti-BRCA1 antibody C20 transrepression activity independently, and the N- (Figure 1a). terminal can bind DNA (Dawson et al., 1995). It has Interaction ofBRCA1 and IFI16 was confirmed by been shown that IFI16 forms stable complexes with coimmunoprecipitation from normally growing HeLa cellular transcription factors such as SP1 and p53 cell nuclear extracts using a novel mouse monoclonal (reviewed in Johnstone and Trapani, 1999; Johnstone antibody (MAb21A8, ref. Okada and Ouchi, 2003) et al., 2000). More recently, the Pyrin domain, which is against the BRCA1 C-terminal and C20 polyclonal commonly found among cell death-associated proteins antibody. BRCA1 was immunoprecipitated from 300 mg such as Pyrin, ASC, and zebrafish caspase and is also ofnuclear extract and samples immunoblotted with referred to as the PAAD/DAPIN domain, has been mouse anti-IFI16 monoclonal antibody 1G7. Three major found in N-terminal IFI16, suggesting a role of IFI16 in IFI16 products were detected in the HeLa nuclear extract the apoptotic pathway (reviewed in Aravind et al., 2001; as a result ofalternative splicing ofthe IFI16 mRNA Fairbrother et al., 2001; Martinon et al., 2001; (Johnstone and Trapani, 1999). Both anti-BRCA1 Pawlowski et al., 2001; Staub et al., 2001). Presumably, antibodies coimmunoprecipitated endogenous IFI16 IFI16 regulates the activity ofcertain transcription (Figure 1b). We tested 12 anti-BRCA1 antibodies, which factors in the nucleus that are involved in the commit- all resulted in BRCA1 and IFI16 coimmunoprecipitation ment to cell death. (data not shown). Reciprocal coimmunoprecipitation In the present study, we further analysed the BRCA1- confirmed the interaction by immunoprecipitation of associated components identified through the mass IFI16 followed by BRCA1 immunoblot (Figure 1c). spectrometric analysis. We identified IFI16 as a pre- Densitometrically, B30% ofBRCA1 bound to B50% viously unknown member ofBASC, and found that ofIFI16. both IFI16 and BRCA1 colocalize in most ofthe cells Next, we mapped the BRCA1-IFI16 binding region examined by confocal laser microscopic analysis. IFI16 by means ofa GST-pulldown assay using 2 mg purified subcellular localization was transiently changed after GST-fusion protein and 500 mg HEK293T total cell DNA damage by IR, suggesting that IFI16 is involved lysate. These results showed that BRCA1 amino acids in the DNA damage pathway. Interestingly, IFI16 distribution, both in the nucleoplasm and nucleolar, is differentially regulated in normal human epithelial cells after DNA damage, but such regulation was abolished in the BRCA1-mutant breast cancer cell line. Most importantly, nucleolar accumulation ofIFI16 was restored by reexpression ofwild-type BRCA1 in BRCA1-mutant cells, strongly suggesting that IFI16 is involved in the BRCA1 pathway activated by DNA damage. Finally, studies using mouse embryonic fibro- blasts (MEFs) obtained from BRCA1-mutant mice with exon 11 deleted or tetracycline-regulatable IFI16 in EJ cells revealed proapoptosis activity ofIFI16 under conditions ofDNA damage, supporting a model of tumor suppression in which the BRCA1/IFI16 complex is involved in the p53-mediated cell death pathway. Results Identification of specific interaction between BRCA1 and IFI16 Our analysis ofproteins coimmunoprecipitating with Figure 1 Specific interaction between IFI16 and BRCA1. (a) BRCA1 identified a BRCA1-associated genome surveil- Schematic diagram ofIFI16 protein structure. The peptide lance complex (BASC) containing proteins ofthe DNA sequence obtained from mass spectrometric analysis of the BASC damage and DNA repair responses such as MSH2/ complex is also shown. (b, c) Coimmunoprecipitation ofendogen- ous BRCA1 and IFI16 from HeLa cell nuclear extract (300 mg). MSH6, MLH1/PMS2, ATM, BLM, replication factor C Total nuclear extract was 60 mg. (d, e) Identification ofthe regions (RFC) and the RAD50-MRE11-NBS1 complex as well where IFI16 and BRCA1 bind to one another. Samples were as p53 and STAT1 transcription factors (Ouchi et al., immunoblotted with the antibodies indicated. (f) HA-tagged full 1998; Zhang et al., 1998; Ouchi et al., 2000; Wang et al., length, but not N-terminal truncated, IFI16 coimmunoprecipitates 2000; Zheng et al.,