A Member of the Pyrin Family, IFI16, Is a Novel BRCA1-Associated Protein Involved in the P53-Mediated Apoptosis Pathway

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 fibroblasts (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., 2000). We identified other proteins with BRCA1 in HEK293T cells. C20 was used for IP of BRCA1. (g) Weak binding ofIFI16 to mutant BRCA1 in HCC1937 cells. with no known function in the DNA damage or DNA DNAseI treatment prior to immunoprecipitation did not affect the repair responses. Among them is the Pyrin domain- interaction. C20 was used for IP of BRCA1

Oncogene IFI16 is a novel BASC inducing p53-mediated cell death JA Aglipay et al 8933 502–802 bound to the Pyrin domain (aa 1–130) ofthe due to UV or IR treatment or hydroxyurea administra- IFI16 N-terminal region (Figure 1d,e). Using HEK293T tion (Scully et al., 1997b). We studied whether DNA cells, interaction ofBRCA1 with the IFI16 N-terminal damage affects IFI16 localization using confocal micro- was confirmed by coimmunoprecipitation with HA- scopy. IFI16 and BRCA1 were detected by anti-IFI16 tagged wt but not N-terminal truncated IFI16 monoclonal antibody 1G7 (green) and anti-BRCA1 (IFI16DN) lackingaa 2–169 (Figure 1f). C-terminal- polyclonal antibody K-18 (red). Normally, BRCA1 truncated BRCA1 in HCC1937 breast cancer cells localization in MCF10A cells is dispersed (Figure 2b). bound weakly to IFI16, indicating that the center region BRCA1 signals slightly decreased 30 min after IR (5 Gy) interacted with IFI16 (Figure 1g). DNaseI treatment of ofDNA damage returned to the basal level after1 h and cell lysates did not affect coimmunoprecipitation, demonstrated a large dot pattern in 3 h (Figure 2f, j, n). suggesting that the proteins reside in the same complex, IFI16 was localized in the nucleoplasm and nucleoli rather than interact through their DNA. Thus, BRCA1 (Figure 2c) and IFI16 colocalization with BRCA1 was physically interacts with IFI16 in normally growing detected throughout the nuclear within 0.5 h after IR cells. (Figure 2d, h). However, following DNA damage, nucleoplasmic IFI16 decreased within 1 h without significant change in nucleolar distribution (Figure 2k), Localization of IFI16 in mammary epithelial cells leading to reduced nucleoplasm colocalization with Previous studies demonstrated subcellular BRCA1 BRCA1 shown by yellow signals in merged images localization changes after DNA damage such as that (Figure 2l). Nucleoplasmic IFI16 reappeared after 3 h and colocalization with BRCA1 was detected as yellow signals where BRCA1 forms a large dot pattern (Figure 2n–p). As shown in Figure 2q, nucleolar localization of IFI16 (green) was confirmed by staining untreated MCF10A cells with antinucleolin (red). IFI16 Western blotting and coimmunoprecipitation indicated transient decrease in BRCA1 expression after IR, with return to original levels after 1 h (Figure 2r, s). Thus, DNA damage determines IFI16 localization in nuclei.

Wild-type BRCA1 is required for relocalization of IFI16 after IR To further study IFI16’s role in BRCA1-associated breast cancer, we explored its localization in the BRCA1 mutant HCC1937 human breast cancer cell line and wt- BRCA1-re-expressing HCC1937 cells (Tomlinson et al., 1998; Scully et al., 1999). IFI16 nucleolar localization was not obvious in parental HCC1937 cells, but a clear nucleolar pattern was detected in wt-BRCA1 reexpressing HCC1937 cells (Figure 3b, B). IFI16 and mutated BRCA1 redistribution in parental HCC1937 cells was not observed 3 h after IR (Figure 3f–o). Signiﬁcantly, in wt-BRCA1-reexpressing HCC1937 cells, IFI16 nucleolar localization increased 1 h after DNA damage with nucleoplasm redistribution within 3 h (Figure 3J, N), a pattern like that in IR-treated MCF10A cells (Figure 2k, o). Reintroduced BRCA1 showed the large dot pattern 3 h after treatment (Figure 3o). As shown in MCF10A cells, colocalization ofboth proteins was detected as yellow signals where BRCA1 forms a large dot pattern (Figure 3p). These results strongly imply IFI16’s Figure 2 BRCA1 and IFI16 colocalize in discrete nuclear foci. involvement in the BRCA1 DNA damage pathway (a, e, i, m): DAPI staining; (b, f, j, n): BRCA1 staining; (c, g, k, o): and the requirement ofwt-BRCA1 protein forits IFI16 staining; and (d, h, l, p): composite BRCA1 and IFI16 staining. Where green and red signals overlap, a yellow pattern redistribution. is seen, indicating colocalization ofBRCA1 and IFI16 (original magniﬁcation, Â 200). (q) Untreated MCF10A cells were stained with anti-IFI16 (green) or antinucleolin (red) antibodies. (r) IFI16 induces apoptosis in BRCA1- and p53-positive cells Western blot analysis ofBRCA1 and IFI16 in response to The IFI16 effect in genotoxic stress-mediated apoptosis IR treatment. IR (5 Gy) induces transient decrease of BRCA1 and IFI16 in MCF10A cells in 0.5 and 1 h, respectively, was examined using BRCA1(À) MEFs according to a (s) Coimmunoprecipitation ofBRCA1 and IFI16 transiently published protocol (Cortez et al., 1999; Xu et al., 2002). decreases 1 h after IR BRCA1(À)/p53(À) MEFs (Xu et al., 2001) were stably

Oncogene IFI16 is a novel BASC inducing p53-mediated cell death JA Aglipay et al 8934

Figure 3 Localization ofIFI16 in HCC1937 cells and wild-type BRCA1-re-expressed HCC1937 cells. Asynchronously growing HCC1937 cells infected with control vector (left panel) or wt-BRCA1 (right panel) were exposed to IR (5 Gy), and were immunoassayed for anti-IFI16 (1G7, green) or anti-BRCA1 (K18, red) antibodies, (a, e, i, m, A, E, I, M): DAPI staining; (b, f, j, n, B, F, J, N): IFI16 staining: (c, g, k, o, C, G, K, O); BRCA1 staining. Re-expression ofwt-BRCA1 restored the nuclear relocalization of IFI16 in response to IR treatment (original magniﬁcation, Â 200)

Figure 4 IFI16 induces cell death ofMEFs expressing BRCA1 and p53 in response to DNA damage. ( a) (top) Stable lines expressing human p53 were generated by infection of MEFs (passage number 1) with retrovirus produced by pBabepuro carrying p53 cDNA. BRCA1(À)/p53(À) MEFs were infected with Ad-BRCA1 or Ad-p53 for 24 h. Expression levels of p53, IFI16 and BRCA1 in MEFs were conﬁrmed and compared with those ofMCF10A cells by immunoblot analysis using DO-1, 1G7 and C20 antibodies (bottom). The indicated MEFs were infected with Ad-IFI16 alone, Ad-BRCA1 alone or both for 24 h then treated by IR. After 12 h, the expression level ofIFI16 or BRCA1 was conﬁrmed by immunoblot analysis, ( b-e) BRCA1(À)/p53(À) or BRCA1(À)/p53( þ ) MEFs (2 Â 105) were infected with the indicated adenoviruses for 24 h, then treated with IR (5 Gy), and the cell viability was examined by trypan blue staining at 24, 36 and 48 h after IR treatment

Oncogene IFI16 is a novel BASC inducing p53-mediated cell death JA Aglipay et al 8935 infected with wt-p53-expressing retrovirus or vector alone, yielding BRCA1(À)/p53(À) and BRCA1(À)/ p53( þ ) MEFs (Figure 4a, upper panel). These MEFs were infected with adenovirus for 24 h and treated with IR (5 Gy). IFI16 and BRCA1 were similarly expressed in coinfected MEFs, resulting in expression levels of these proteins similar to those ofMCF10A normal epithelial cells (Figure 4a, upper and lower panels). Cell viability was determined by trypan blue exclusion. Neither signiﬁcant apoptosis nor inhibition ofcell growth was detected in BRCA1(À)/p53(À) MEFs and IR-untreated BRCA1(À)/p53( þ ) MEFs after expressing the indicated proteins (Figure 4b–d). In contrast, BRCA1(À)/p53( þ ) MEFs varied phenotypes after IR treatment according to the proteins transduced (Figure 4e): Expression ofLacZ-, IFI16- or IFI16 DN deﬁcient in binding to BRCA1 slightly retarded cell growth, although no obvious apoptosis was detected. Furthermore, expression ofBRCA1 alone or BRCA1 plus IFI16DN resulted in resistance to growth retardation induced by IR treatment. Importantly, coexpression ofBRCA1 and IFI16 in BRCA1( À)/p53( þ ) MEFs led to severe retardation ofcell growth and loss ofcell viability 36–48 h after IR treatment. These results suggest that IFI16 plays roles in negative regulation of cell growth under conditions ofDNA damage when BRCA1 and p53 are coexpressed.

IFI16 collaborates with p53 to induce apoptosis We further explored the mechanism of apoptosis using tetracycline (tet)-regulated IFI16 in p53-negative human bladder EJ cells (EJ-IFI16), in which IFI16 was induced by removing tet from cell culture (Figure 5a). When tet was removed, cells were infected with the adenovirus indicated in Figure 5b for 24 h. Cells were then treated with actinomycin D (ActD) for 12 h before FACS analysis. Without IFI16 induction (tet( þ )), infection with GFP or p53 did not induce apoptosis, which was measured on the basis ofthe subG1 fraction. When IFI16 was induced (tet(À)), p53 infection strongly Figure 5 IFI16 induces p53-mediated apoptosis. Regulation of induced apoptosis after ActD treatment although p53 IFI16 expression in EJ-IFI16 cells at 6, 12, 24 and 36 h after tet- alone did not lead to an increased subG1 fraction. These removal. When tet was removed, cells were infected with Ad-GFP or Ad-p53 for 24 h. Cells were treated with ActD (10 mg/ml) for 12 h results indicate that IFI16 collaborates with p53 and cell cycle proﬁles were analysed by FACS. Populations ofcells activated by a genotoxic reagent to induce apoptosis. in the subG1 fraction are indicated

Discussion showing the redistribution ofIFI16 afterDNA damage Mass spectrometric analysis is a well-established tech- and the induction ofapoptosis in cells with DNA nique for identifying specific protein–protein interac- damage suggest that IFI16 is a functional component of tion. Previously, we identified several DNA damage BASC. Furthermore, we discovered proapoptosis activ- repair proteins in BASC – MSH2, MSH6, MLH1, ity ofIFI16 in cells expressing both wild-type p53 and ATM, BLM and the RAD50-Mre11-NBS1 complex BRCA1. (Wang et al., 2000). However, BRCA1 protein has been Recent studies applying a profile-to-profile alignment found to reside in a large protein complex with >2 MDa algorithm to a library ofapoptosis-associated proteins strongly suggesting that BASC contains many other identified a novel motifcalled the Pyrin/PAAD/DAPIN components. We added IFI16 as a new member to the domain (reviewed in Aravind et al., 2001; Fairbrother set ofBRCA1-interacting proteins; the specific interac- et al., 2001; Martinon et al., 2001; Pawlowski et al., tion between BRCA1 and IFI16 was revealed by 2001; Staub et al., 2001). This new class ofdeath domain coimmunoprecipitation experiments. The present data is found in Pyrin, zebrafish caspase, ASC (apoptosis

Oncogene IFI16 is a novel BASC inducing p53-mediated cell death JA Aglipay et al 8936 spec protein), IFI16 and AIM2, suggesting that the DMEM-10% fetal bovine serum (FBS), or RPMI1640-10% proteins in question are involved in cell death pathway. FBS (HCC1937 cells). EJ-tet cells were maintained in the Our studies provided evidence that the Pyrin domain of presence oftetracycline (1 mg/ml). Cell extracts were prepared IFI16 is a protein–protein interaction module that is in EBC buffer (50 mM Tris, pH 8, 120 mM NaCl, 0.5% important for binding to BRCA1. Nonidet P-40 [NP-40]), with the addition of50 m M NaF, 1 mM sodium orthovanadate, 100 mg/ml polymethylsulfonyl fluoride Confocal laser microscopic images revealed that (PMSF), 20 mg/ml aprotinin and 10 mg/ml leupeptin. Nuclear IFI16 coexists with BRCA1 predominantly in the nuclei extracts ofgrowing HeLa cells were prepared followinga in normal mammary epithelial cells. Similar IFI16 published protocol (Gu et al., 1999). Ionizing radiation was localization was observed in human normal diploid administrated by means ofMARK2 IRRADIATOR (JL fibroblasts (data not shown). Interestingly, only nucleo- Stephen & Associate), and actinomycin D (ActD) was plasmic IFI16 dramatically decreased 1 h after damage purchased from Sigma. in MCF10A cells whereas the concentration ofnucleolar IFI16 was unaffected. Since FACS analysis revealed Construction of BRCA1 and IFI16 expression vectors that the cell cycle profile showed neither cell cycle arrest nor apoptosis within 1 h after IR (data not shown), it is GST fusion proteins expressing six segments covering full- length BRCA1 have been described (Scully et al., 1997a). Full- unlikely that relocalization ofIFI16 results fromcell length IFI16 cDNA was PCR amplified using following cycle arrest or apoptosis. Although the mechanism by primers: (#1) 50-AAAGGATCCATGTCTGTAAAGATGG- which nuclear IFI16 localization is regulated remains to GAAAAAAATAC-30 and (#6) 50-AGCGGCCGCTTAGAA- be clarified, it is conceivable that DNA damage- GAAAAAGTCTGGTGAAGTTTC-30 from a HeLa cells activated kinases such as ATM, ATR, Chk2 and cDNA library (a gift from Dr H Nojima, Osaka University, DNA-PK play a role in determining IFI16 distribution. Japan). Three segments ofIFI 16 cDNAs were PCR amplified IFI16 contains at least four potential phosphorylation by primer (#1) and primer (#2) 50-CTGAGCTCCAGGA consensus motifs (S/TQ) for ATM kinase, namely, at GTTGCCTCTGCTCC-30, primer (#3) 50-AAAGGATCCAG AAATGTTCTCA AAAAGCCCAGTGATA-30 and primer positions 153, 239, 241 and 628. 0 HCC1937 breast cancer cells demonstrated a different (#4) 5 -AGCGGCCGCTGACATCTGGTTCTTTTTTCTAA GTCG-30, primer (#5) 50-AAAGGATCCAATGACCCCAA pattern ofIFI16 relocalization after DNA damage. GAGCATGAAGCTACCCCAG-30 and primer (#6). BamHI– Thus, although IFI16 was nuclear in HCC1937 cells, no SacIorBamHI–NotI fragments amplified by these primers nucleolar accumulation was observed. The requirement were subcloned into pCRScript (Stratagene), and the DNA of BRCA1 for IFI16 relocalization after DNA damage sequence was confirmed. Amplified fragments were transferred was demonstrated in wild-type BRCA1-reintroduced to pGEX4T vector (Pharmacia). Synthesis ofGST fusion HCC1937 cells. Considering that mutant BRCA1 in proteins and their partial purification on glutathione-sephar- HCC1937 cells is differently phosphorylated under ose beads were performed as described (Kaelin et al., 1991). conditions ofDNA damage than the wild-type protein (Okada and Ouchi, 2003), the BRCA1 phosphorylation Generation of adenovirus status may play a role in determining the localization of In this protocol (Ouchi et al., 2000), briefly, cDNAs ofGFP, the complex containing BRCA1-associated proteins. IFI16 and p53 was subcloned into pShuttle-CMV, and It has been shown that BRCA1 increases p53 stability recombination with adenovirus genomic DNA was carried in a p19ARF-dependent manner (Ouchi, T, unpublished out in BJ5180 bacterial cells. Isolated recombinant viruses observation; ref. Somasundaram et al., 1999). In our were amplified in HEK293 cells and used for infection of cells investigation of whether IFI16 also affects the p53 at multiplicity ofinfection(m.o.i.) of5 or 50, respectively. protein level, no significant increase was observed in Adenovirus BRCA1 was described previously (Ouchi et al., immunoblot analysis (data not shown). The mechanism 2000). ofapoptosis induced by IFI16 is currently under investigation. In this connection, it is striking that Western blot analysis, immunoprecipitation and GST-pulldown AIM2, one ofthe proteins containing the Pyrin/PAAD/ assay DAPIN domain (Aravind et al., 2001), is a tumor Whole cell extract (20 mg) was loaded per lane by 6% SDS– suppressor protein ofhuman melanoma (Absent In polyacrylamide gel electrophoresis (SDS–PAGE). Transfer Melanoma, DeYoung et al., 1997). Among proteins onto nitrocellulose was performed using a semidry transfer containing this domain, loss ofapoptosis regulation by method (TRANS-BLOT, BIO-RAD), in 50 mM Tris base, the Pyrin domain is likely to be closely associated with 40 mM glycine, 0.37 g/l SDS, 20% methanol (for 3 h at 15 V). tumor malignancy. After blocking with 1% nonfat dried milk in TBS-T (20 mM Tris, pH 8, 0.9% NaCl, 0.05% Tween-20), the primary antibody, Ab-1 for BRCA1 (Oncogene Science) or 1G7 for IFI16 (Santa Cruz), was used at 2 mg/ml in PBS/1% nonfat Materials and methods dried milk, for 1 h at room temperature. The secondary antibody was peroxidase-conjugated goat anti-mouse IgG Cell culture and preparation of total cell and nuclear extracts (H þ L, Jackson Immunoresearch), at 1 : 10 000 in 1% nonfat MCF10A and HCC1937 cells were obtained from ATCC. milk/TBS-T. Signals were developed by ECL (Amersham). IP BRCA1-re-expressed HCC1937 cells were described previously ofBRCA1 was performedas described (Ouchi et al., 2000). (Scully et al., 1999). Tetracycline-regulatable IFI16 in EJ Monoclonal antibody 21A8 was generated by immunizing human bladder carcinoma cells were established following mice with GST-BRCA1 (aa l314–1863) as described (Okada published protocols (Lee et al., 2000). Cells were cultured in and Ouchi, 2003). The GST-pulldown assay was performed

Oncogene IFI16 is a novel BASC inducing p53-mediated cell death JA Aglipay et al 8937 using total cell lysates ofHEK293 cells as described (Ouchi retrovirus produced by pBabepuro carrying p53 cDNA and et al., 1998, 2000). BOSC23 packaging cells (Pear et al., 1993) after puromycin selection (7 days, 1.5 mg/ml). These MEFs (2 Â 105) were Immunostaining analysis further infected with the indicated adenoviruses, with or without IR (5 Gy). Cell survival assay using BRCA1-mutant Cells were fixed for 1 h in phosphate-buffered saline (PBS)-3% MEFs were studied by trypan blue staining using a published paraformaldehyde/2% sucrose solution, followed by 5 min protocol (Cortez et al., 1999; Xu et al., 2002). permeabilization at room temperature in Triton buffer (0.5% Triton X-100, 20 mM HEPES, 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose). For blocking 5% normal horse serum/5% normal goat serum was used. BRCA1, IFI16 and nucleolin Abbreviations were visualized using rabbit polyclonal antibody K-18, BASC, BRCA1-associated genome surveillance complex; monoclonal 1G7 antibody and rabbit polyclonal antibodoy MEF, mouse embryonic fibroblast; HEK293, human embryo- C23 (all from Santa Cruz), respectively. All secondary nic kidney 293; IR, ionizing radiation. antibodies used were species-specific fluorochrome-conjugated antibodies from Jackson Immunoresearch (Texas Red-X for mouse IgG and fluorescein isothiocyanate [FITC] for rabbit Acknowledgements IgG), used at 1 : 100 throughout. Nuclei were stained with We are grateful to Drs David M Livingston and George R DAPI. All images were collected by the Leica TCS-SP Stark for helpful discussion, Ralph Scully for HCC1937- Confocal Laser Scanning Microscope and processed using BRCA1 cells, Mutsuko Ouchi for generating and characteriz- Adobe Photoshop software. ing anti-BRCA1 monoclonal antibodies, Dr Scott Henderson, Jane Sue and Bryan Kloos for confocal laser microscope analysis, and the Mount Sinai School ofMedicine Core Flow cytometry and cell survival assay Facilities for the monoclonal antibody, DNA sequencing and For cell cycle analysis ofEJ-tetIFI16 cells, tet-removal cells microscopic analysis. This work was supported by an NIH were infected with adenovirus for 24 h and treated with ActD Predoctoral Training Award (JAA), the New York City for 12 h. Cells undergoing apoptosis were analysed using Council Speaker’s Fund (TO), an EMPIRE Grant ofState FACSCalibur (Beckton and Dickinson). MEFs were described ofNew York (TO) and National Cancer Institute Awards elsewhere (Xu et al., 2001). Stable lines expressing human p53 CA80058, CA78356 (SWL), CA84199 (JQ) and CA79892 and were generated by infection of MEFs (passage number 1) with CA90631 (TO).

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Oncogene