Inhibition of NF-␬B by ZAS3, a -finger that also binds to the ␬B motif

Joung-Woo Hong*, Carl E. Allen†, and Lai-Chu Wu*‡§

*Ohio State Biochemistry Program and ‡Departments of Molecular and Cellular Biochemistry and Internal Medicine, Ohio State University, Columbus, OH 43210; and †Department of Pediatrics, Columbus Children’s Hospital Research Institute, Columbus, OH 43205

Edited by George R. Stark, Cleveland Clinic Foundation, Cleveland, OH, and approved August 20, 2003 (received for review May 20, 2003) The ZAS are large zinc-finger transcriptional proteins (9, 10). This observation has prompted several groups, including implicated in growth, , and lymphoid develop- ours, to investigate whether the ZAS proteins are non-Rel-␬Bs. ment. Recombinant ZAS fusion proteins containing one of the two Results of antibody gel-shift assays and reporter assays DNA-binding domains have been shown to bind specifically to the suggest that non-Rel-␬Bs are related to ZAS2 and -3 (11–13). ␬B motif, but the endogenous ZAS proteins or their physiological However, another study suggests that non-Rel-␬B may be related functions are largely unknown. The ␬B motif, GGGACTTTCC, is to Sp1 (14). Recently, several immortalized ZAS3Ϫ/Ϫ preB cell a gene regulatory element found in promoters and enhancers lines have been established from a malignant teratoma that of involved in immunity, inflammation, and growth. The developed spontaneously in a ZAS3Ϫ/ϪRAG2Ϫ/Ϫ chimeric Rel family of NF-␬B, predominantly p65.p50 and p50.p50, are mouse (15). To clarify the origin of non-Rel-␬B, we examine the factors well known for inducing gene expression by ␬B-binding species and show that non-Rel-␬B is absent in those Ϫ Ϫ means of interaction with the ␬B motif during acute-phase re- ZAS3 / cells. Additionally, in keeping with our previous re- sponses. A functional link between ZAS and NF-␬B, two distinct sults, which show that ZAS3 associates with TRAF2 to inhibit Ϫ Ϫ families of ␬B-binding proteins, stems from our previous in vitro NF-␬B activation, NF-␬B is constitutively expressed in ZAS3 / studies that show that a representative member, ZAS3, associates cells. Because ZAS3 can regulate ␬B-mediated transcription and with TRAF2, an adaptor molecule in tumor necrosis factor signal- influence the activity of NF-␬B, this mechanism provides a ing, to inhibit NF-␬B activation. Biochemical and genetic evidence checkpoint to control the reprogramming of gene expression. presented herein shows that ZAS3 encodes major ␬B-binding proteins in B lymphocytes, and that NF-␬B is constitutively acti- Materials and Methods vated in ZAS3-deficient B cells. The data suggest that ZAS3 plays Plasmids. The reporter plasmids, 3ϫ␬B-Luc and p7ϫ␬B, and crucial functions in maintaining cellular homeostasis, at least in expression constructs for p50 and p65 have been described (16, part by inhibiting NF-␬B by means of three mechanisms: inhibition 17). Both plasmids were used in reporter gene assays and yielded of nuclear translocation of p65, competition for ␬B gene regulatory similar results. For the construction of a ZAS3 expression vector, elements, and repression of target gene transcription. a 5.7-kb XhoI ZAS3 cDNA fragment (nucleotides 973–6693) was inserted into the XhoI site of the expression vector pCMV-Tag2 he Rel family of NF-␬B encodes important transcription (Stratagene). pSG-ZASC was constructed by inserting a 1.6-kb Tfactors that regulate the induction of genes involved in ZAS3 cDNA fragment (nucleotides 5831–7490) into the EcoRI immunological, inflammatory, and antiapoptotic responses (1, site of the plasmid pSG424 (18). 2). In most resting cells, NF-␬B is sequestered in the cytoplasm, Antibodies. ␬ bound to a family of inhibitory molecules, inhibitor (I)␬B. Many ZAS3 antisera have been described (13). NF- B antibodies were gifts from Denis Guttridge, Ohio State Univer- stimuli, including lipopolysaccharide (LPS) (3) and inflamma- sity: p65 (Rockland, Gilbertsville, PA), p50 (Upstate Biotech- tory cytokine tumor necrosis factor (TNF) (4), activate the nology, Lake Placid, NY), p52 (K-27; Santa Cruz Biotechnolo- NF-␬B signal transduction pathway and lead to the phosphor- gy), and c-Rel (N; Santa Cruz Biotechnology). Antibodies for ylation and degradation of I␬B. NF-␬B is then imported into the I␬B␣ (C-21), p-I␬B␣ (B-9), hsp90, TRAF1, TRAF2, histone nucleus, where it activates transcription of target genes. In many H1 (Santa Cruz Biotechnology), and FLAG (Sigma) were cell types, the predominant NF-␬B species are p65.p50 and purchased. p50.p50. However, other ␬B-DNA-binding proteins (designated as non-Rel-␬Bs) distinct from NF-␬B have been characterized. Cell Lines and Transient Transfection Assays. Immortalized Generally, non-Rel-␬Bs differ from NF-␬B by size, immunoge- Ϫ Ϫ Ϫ Ϫ ZAS3 / B1 and ZAS3 / B2 preB cells were previously called nicity, sensitivity to detergents, and sequence specificity to the KRC-mIII and -mIV, respectively (15). PreB cells were cultured IMMUNOLOGY ␬B motif (Table 1). in complete medium (RPMI medium 1640) containing 10% FCS An additional gene family known to encode proteins that also ϩ ϩ Ϫ Ϫ and 50 ␮M 2-mercaptoethanol. p65 / and p65 / mouse em- bind to the ␬B motif is called the ZAS family (5). ‘‘ZAS’’ bryonic fibroblasts (MEF), human embryonic kidney (HEK) describes a structural domain that contains a pair of C2H2 zinc ͞ 293, and Cos-7 cells were cultured in DMEM supplemented with fingers, an acidic region, and a serine threonine-rich sequence 10% FCS. For transient transfection, 2–5 ϫ 105 suspension cells (6). There are three ZAS proteins, ZAS1, -2, and -3, in mammals were seeded into each well of a 24-well plate and were cultured and a distant relative, schnurri (shn), in Drosophila. The ZAS overnight in complete medium. After 24 h, reporter plasmid (250 proteins have been shown to regulate transcription of genes ng), with or without ZAS3 expression plasmid, was transfected involved in immunity, development, and metastasis (5). They into cells by using the cationic reagent DMRIE-C (2 ␮l per have also been shown to regulate signal transduction. Schnurri associates with Mad͞Smad to modulate decapentaplegic͞ transforming growth factor ␤ signaling during embryonic This paper was submitted directly (Track II) to the PNAS office. development (7). ZAS3 associates with TRAF2 to inhibit Abbreviations: TNF, tumor necrosis factor; LPS, lipopolysaccharide; EMSA, electrophoretic TNF-induced NF-␬B-dependent transactivation and JNK mobility-shift assay; MEF, mouse embryonic fibroblast; I␬B, inhibitor ␬B; IKK, I␬B kinase; phosphorylation in vitro (8). HEK, human embryonic kidney. Extensive DNA–protein interaction analyses have shown that §To whom correspondence should be addressed. E-mail: [email protected]. individual ZAS domains bind specifically to ␬B-like sequences © 2003 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.2133048100 PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12301–12306 Downloaded by guest on September 25, 2021 Table 1. Distribution and properties of non-Rel-␬B Name Distribution Properties Ref.

Band A BU-11 (pro-B to early pre-B), WEHI-231 125–135 kDa; did not react with NF-␬B antibodies; 20 (sIgϩ), and 70Z͞3 (late pre-B) decreased by polycyclic aromatic hydrocarbons 110- and 180-kDa species Jurkat T lymphocyte Differential specificity of DNA binding for various ␬B 35 enhancer sequences from NF-␬B; induced by phorbol 12-myristate 13-acetate Abnormal ␬B-binding protein S107 plasmocytoma Localize in cytoplasm; binding abolished by deoxycholate 19 and NP-40; 110–115 kDa; suggested to inhibit nuclear translocation of cytoplasmic NF-␬B and thereby void ␬B-mediated expression Brain-specific ␬B-binding protein Primary neurons Does not activate ␬B-mediated expression; reacts with 11, 36 (BETA) antisera against ZAS2 but not ZAS1 or p65 antibodies; DNA binding depends on zinc ion Neuronal ␬B-binding factor (NKBF) Brain, primary neurons, cerebellum, Does not react with NF-␬B antibodies (p50, p65, c-Rel, 12 and cell lines (N9, CBL, and NT2) p52, and RelB) or with ZAS2 antibodies; sensitive to DOC; present in p105͞p50 knockout mice; composed of proteins of 27, 82, and 109 kDa; induces by 8-Br-cGMP; inhibits by glutamate Developing-brain factors 1 and 2 Developing cortex, brain not in lung, 110 and 115 kDa; sensitive to DOC; binding-site 37 (DBF1 and -2) liver, intestine, heart, and kidney specificity similar to NF-␬B; does not react with p100 or p105 antibodies; developmentally regulated in brain Posthepatectomy factor (PHF) Regenerating livers Induced Ͼ1,000-fold within minutes postheptatectomy in 38 a protein-synthesis-independent manner; DNA binding sensitive to phosphatases Not named 293 cells Induced by NF-␬B-activating kinase 39

well) (Invitrogen). Plasmid pCH110 (250 ng) encoding ␤-galac- present in preB cells, in agreement with previous studies of B cell tosidase was included to normalize transfection efficiency. lineages (19, 20). The data also show that non-Rel-␬B is immu- pCMV-Tag2 was supplemented to yield a total plasmid concen- nologically related to ZAS3. tration of 1 ␮g in each transfection. Thirty-six hours posttrans- To clarify the origin of the non-Rel-␬B in B lymphocytes, fection, LPS (10 ␮g͞ml) was added to a subset of cells, and all EMSA was performed by using a genetic background where cells were harvested 4 h later. For adherent cells, reporter ZAS3 was knocked out by homologous recombination. plasmid (10 ng), p65 and p50 expression plasmids (10 ng each), ZAS3Ϫ/ϪB1 and ZAS3Ϫ/ϪB2 were preB cell lines independently ␮ ␮ Ϫ Ϫ Ϫ Ϫ ZAS3 expression plasmid (0–0.4 g), lipofectamine 2000 (2 l derived from a malignant teratoma of a RAG2 / ZAS3 / per well; Invitrogen), and pCMV-Tag2 (to adjust total DNA to chimera mouse (15). In EMSA, a prominent p65.p50 complex ␮ ␤ 0.5 g) were used in each transfection. Luciferase and -galac- but not the C1 complex was observed from either nuclear tosidase activity were measured as described (15). extracts of the ZAS3Ϫ/Ϫ cells (Fig. 1 B and C). Previous extensive RNA studies, including Northern blot analysis, RT-PCR, RNase Electrophoretic Mobility-Shift Assays (EMSAs), Antibody-Supershift Assays, and Immunoblot Analyses. EMSAs and antibody-supershift assays, with a 32P-labeled probe (2 ϫ 104 cpm) containing a ␬B site (underlined), 5Ј-CCGGGGGGACTTTCCGCTCCAC-3Ј, were performed as described (10). Immunoblot analyses were performed as described (15). The presence of equivalent protein loading was verified by protein staining of duplicated gels or by incubating duplicated protein blots with histone H1 or heat- shock protein hsp90 antibodies. Results and Discussion ZAS3 Antisera React with a Non-Rel-␬B Absent in ZAS3؊/؊ Cells. In EMSA, nuclear extracts prepared from a standard preB cell line, 38B9, and a 32P-␬B probe yielded several sequence-specific DNA–protein complexes (Fig. 1A, lane 1). The prominent complexes were designated C1, -2, and -3. The addition of a ZAS3-antiserum but not preimmune serum to the binding reactions diminished the amount of C1 (Fig. 1A, lanes 2 and 3). Fig. 1. NF-␬B and ZAS3 are the major ␬B DNA-binding proteins in B Antibodies against p65 and p50 also altered the pattern of the lymphocytes. (A) EMSA of 32P-␬B oligonucleotide and 38B9 nuclear ex- DNA–protein complexes (Fig. 1A, lanes 4 and 5). Likely, C2 and tracts. Antibodies or unlabeled DNA (20 ng; 100-fold excess) supplemented -3 were composed of p50.p50 and p65.p50, respectively. The to binding reactions are indicated at the top of each lane. The major DNA–protein complexes were designated as C1, -2, and -3. (B) EMSA of specificity of the protein–DNA interaction was demonstrated by 32 ␬ ␬ P- B oligonucleotide and nuclear extracts. Lane 1, 38B9; lanes 2 and 3, DNA competition. Unlabeled B oligonucleotides (20 ng; 100- representative ZAS3-deficient cells ZAS3Ϫ/ϪB1 and ZAS3Ϫ/ϪB2. (C) Gel su- fold excess) competed away the formation of all complexes (Fig. pershift assays show the presence of p65 and p50 in the predominant 1A, lane 7), whereas a similar amount of Sp1 oligonucleotide was ␬B–DNA protein complex derived from ZAS3Ϫ/ϪB1 nuclear extracts. Trian- noncompetitive (Fig. 1A, lane 6). Therefore, non-Rel-␬Bis gles indicate antibody supershifted complexes.

12302 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2133048100 Hong et al. Downloaded by guest on September 25, 2021 nuclear extracts. The higher NF-␬B-binding activity from the nucleus of ZAS3Ϫ/Ϫ cells was due, at least in part, to constitutive nuclear localization. Immunoblot analysis of nuclear extracts showed that the amount of p65 in ZAS3Ϫ/Ϫ cells was 5- to 10-fold higher than in 38B9 cells (Fig. 2B), in keeping with EMSA results. In addition, LPS (10 ␮g͞ml for 4 h) increased the nuclear levels of p65 significantly in 38B9 cells but minimally in ZAS3Ϫ/Ϫ cells. The constitutive expression of nuclear p65 in ZAS3Ϫ/Ϫ cells and the induction of p65 in 38B9 cells by LPS were specific, because the amounts of p50 and histone H1 were comparable among all samples. Reporter gene assays were performed to assess the ␬B- mediated transcriptional competence of the preB cells. In line with the increase of ␬B-DNA binding and nuclear p65, the ␬B-reporter activity was 30-fold higher from ZAS3Ϫ/Ϫ than 38B9 cells (Fig. 2C). This observation agrees with the fact that, although all NF-␬B contain a for DNA binding, only p65, RelB, and c-Rel contain transactivation domains (1). In addition, LPS (10 ␮g͞ml for 4 h) augmented ␬B-reporter activity 5- to 8-fold in 38B9 cells but minimally in ZAS3Ϫ/Ϫ cells. The increase in NF-␬B binding and consequently ␬B-mediated transcription in ZAS3Ϫ/Ϫ cells was due, in part, to the up-regulation of nuclear p65. Fig. 2. Constitutive activation of NF-␬B in ZAS3Ϫ/Ϫ cells. (A) EMSA of 32P-␬B The remarkable effect of ZAS3 on the ␬B-reporter suggests oligonucleotide, nuclear extracts (N), or cytoplasmic extracts (C), and with (ϩ) that ZAS3 should have a major impact on NF-␬B-dependent or without (Ϫ) LPS (10 ␮g͞ml for 4 h). (B) Immunoblot analysis of nuclear gene expression and cell fate. Accordingly, ZAS3 had been extracts. (C) Reporter gene assays. Indicated cells were cotransfected with shown to control the expression of TNF␣,anNF-␬B target p7ϫ␬B (0.25 ␮g) and pCH110 (0.1 ␮g). Thirty-six hours posttransfection, ␮ ͞ gene, in macrophages and in an epithelial cell line (8). duplicated cell samples were incubated with LPS (10 g ml, 4 h). Luciferase Additionally, tumorogenesis and immortalization had been activity, normalized to ␤-galactosidase activity, from unstimulated 38B9 cells was assigned as ϩ1. (D and E) Immunoblot analyses of cytoplasmic extracts. observed in ZAS3-deficient animals and cells, respectively (15). In keeping with the differential expression of NF-␬B, immunoblot analyses showed that the amount of TRAF1 and -2, whose expression was controlled by NF-␬B, was higher in protection, and in situ hybridization, had shown that ZAS3 was Ϫ Ϫ expressed specifically in neuronal and lymphoid tissues (6, 10, ZAS3 / cells than in 38B9 cells (Fig. 2D). ␬ ␬ 21–23). Therefore, the lymphoid expression, immunogenicity, In most cell types, NF- B associated with I B in the cytoplasm ␬ and ␬B-binding capability of ZAS3 and non-Rel-␬B are similar. but was translocated into the nucleus when I B was degraded. To ␬ further characterize NF-␬B signaling in ZAS3Ϫ/Ϫ cells, the Because non-Rel- B had been detected in different lineages of ␬ ␣ B cells (20), the absence of C1 from ZAS3Ϫ/Ϫ B cells supports expression and phosphorylation status of I B were examined. ␬ The amount of I␬B␣ in unstimulated 38B9 cells was significantly our notion that non-Rel- B was a product of ZAS3. Although the Ϫ/Ϫ ZASϪ/Ϫ cell lines have been classified as preB cells based on higher than that of ZAS3 cells (Fig. 2E), in keeping with the constitutive expression of NF-␬B in the latter cells. Of interest, morphology and cell-surface markers (15), there are no isogenic Ϫ/Ϫ Ϫ/Ϫ ϩ/ϩ I␬B␣ was hyperphosphorylated in ZAS3 cells, suggesting an controls for those cells. The ZAS3 and ZAS3 cells studied ␬ Ϫ/Ϫ inevitably have other intrinsic genetic and subtle developmental induction of I B kinase (IKK). Taken together, in ZAS3 cells, the absence of ZAS3, a TRAF2 inhibitor, activates the assembly differences. However, similar differential NF-␬B regulation had Ϫ Ϫ of the IKK complex. IKK phosphorylates I␬B, which marks I␬B been observed in two ZAS / preB cells and several standard ϩ ϩ for ubiquitination and degradation. NF-␬B then translocates into ZAS3 / preB cell lines examined (38B9, 22D6, and IIA1.6). Ϫ Ϫ ϩ ϩ the nucleus, leading to activation of gene expression. The establishment of isogenic ZAS3 / and ZAS3 / mice will allow the performance of physiologically relevant experiments ZAS3 Directly Represses NF-␬B-Mediated Transcription. ZAS3 ex- and the investigation of how ZAS3 deficiency may affect TNF- pression was restored in ZAS3Ϫ/Ϫ cells to demonstrate that the induced activation of NF-␬B and JNK in an animal model. ␬ induction of NF- B was a consequence of ZAS3 deficiency. To IMMUNOLOGY this end, a 7.2-kb ZAS3 cDNA harboring the largest ORF of Constitutive Expression of p65 in the Nucleus of ZAS3-Deficient Cells. 2,348 residues was constructed by conventional molecular clon- Previous in vitro studies have implicated ZAS3 in the inhibition ing procedures by using clones isolated from standard cDNA or of TNF-driven NF-␬B activation by sequestering an adaptor ␬ genomic libraries. That DNA fragment, when inserted in-frame molecule, TRAF2, essential for the assembly of the I B kinase into the FLAG-tag expression plasmid pCMV-Tag2, yielded low complex (8). The prominence of p65.p50 in nuclear extracts of expression in HEK 293 cells (data not shown). Subsequently, Ϫ/Ϫ ZAS3 cells supports that model. To demonstrate a functional plasmids with shorter inserts were constructed, and a DNA ␬ ␬ link between ZAS3 and NF- B, NF- B was characterized in fragment encoding amino acids 106–2013 of ZAS3, which con- ZAS3Ϫ/Ϫ and ZAS3ϩ/ϩ cells by EMSA. In unstimulated Ϫ Ϫ tained all known structural features of ZAS3, yielded abundant ZAS3 / cells, NF-␬B-binding activity was mostly found in fusion proteins (Fig. 3A). The FLAG-ZAS3 fusion protein was nuclear extracts (Fig. 2A). As such, LPS (10 ␮g͞ml for 4 h), a detectable from nuclear but not from cytoplasmic extracts. reagent commonly used to stimulate NF-␬B in B cells (3), Similar cellular distribution was observed for a nuclear protein showed minimal effect. Conversely, NF-␬B DNA-binding activ- control, histone H1. Previously, ZAS3 antibodies detected sig- ity was found in both cytoplasmic and nuclear extracts from 38B9 nals from the nucleus and cytoplasm (8). The cytosolic signal cells. On LPS stimulation, NF-␬B-binding activity from 38B9 might be derived from smaller ZAS3 isoforms generated by cells was reduced to minimal levels in the cytoplasmic extracts alternative splicing (22). with a concomitant increase of NF-␬B-binding activity in the Reporter gene assays initially showed that ZAS3 expression

Hong et al. PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12303 Downloaded by guest on September 25, 2021 Fig. 4. ZAS3 inhibits nuclear localization of p65. Immunoblot analysis of 38B9 and ZAS3Ϫ/Ϫ cells after transient transfection with FLAG-ZAS3 (ϩ)or parental FLAG (Ϫ) expression plasmids showed that p65 was localized mainly Fig. 3. ZAS3 represses NF-␬B-mediated transcription. (A) Expression of in the cytoplasmic extracts (CE) of 38B9, but in the nuclear extracts (NE) of recombinant FLAG-ZAS3 proteins in HEK 293 cells. (Upper) Structural domains ZAS3Ϫ/Ϫ cells. ZAS3 expression in both cells reduced the amount of nuclear p65 of recombinant FLAG-ZAS3 protein. NLS, nuclear localization signal. (Lower) with a concomitant increase of cytoplasmic p65. The second p65 was an Immunoblot analyses of HEK 293 cells transiently transfection with FLAG-ZAS3 overexposure of the first to highlight the reduction of nuclear p65 by ZAS3 expression plasmid or parental plasmid, pCMV-Tag2 (FLAG). N, nuclear ex- expression in 38B9 cells. As controls for protein loading and cell fractionation, tracts; C, cytoplasmic extracts. (B) Reporter gene assays. Plasmids 3ϫ␬B-Luc (10 immunoblot analyses were also performed with a nuclear protein control, ng) and pCH110 (10 ng) were cotransfected with the control parental vector histone H1, and a cytosolic protein control, heat-shock protein 90 (hsp90). (pCMV-Tag2; white bar) or indicated amount of FLAG-ZAS3 expression plas- mid (black bars) in HEK 293 cells. The normalized luciferase activity of cells cotransfected with the control vector was assigned to 100%. (C) ZAS3 re- association, and transcriptional activity of p65 (24, 25). However, ␬ pressed NF- B-activated transcription. Transient transfection experiments incubation of the samples with shrimp alkaline phosphatases had were performed with (ϩ) or without (Ϫ) p65 and p50 expression plasmids (10 ng each) in HEK 293 cells. The normalized luciferase activity of cells transfected no effect on the gel mobility of p65 (data not shown), suggesting with p65 and p50 expression plasmids was assigned as 100. (D) ZAS3 repressed the difference might not be due to protein phosphorylation. a ␬B-reporter gene in ZAS3Ϫ/Ϫ cells. Normalized luciferase activity of trans- Previously, two such closely migrating p65-antibody reacting fection in 38B9 cells with the reporter gene only was assigned as 1. species were observed in immunoblot analysis of rat livers, and treatment with calf intestine alkaline phosphatases also did not resolve the difference in their gel mobility (26). reduced the activity of ␬B-reporter genes in HEK 293 (Fig. 3B) and 38B9 cells (data not shown). Subsequent cotransfection ZAS3 Represses Transcription Independent of p65. Although p65 experiments with ZAS3 and NF-␬B (p50 and p65) expression contains the transactivation domain, the activity of the ␬B- constructs showed that ZAS3 repressed NF-␬B-mediated reporter genes was not directly proportional to the amount of transactivation of the ␬B reporter (Fig. 3C). Significantly, p65. For example, whereas the amount of p65 in LPS- Ϫ Ϫ ZAS3 expression also inhibited the ␬B reporter in ZAS3 / stimulated 38B9 was more than that in ZAS3Ϫ/Ϫ cells (compare cells (Fig. 3D), providing a link between ZAS3 deficiency and Fig. 2B, lanes 2 and 4), the ␬B reporters were 5-fold more NF-␬B activation. active in the latter (Fig. 2C). There are several not mutually To evaluate how ZAS3 expression represses NF-␬B- exclusive possibilities to account for this discrepancy. First, dependent transactivation, the level of NF-␬B in the preB cells RelB and c-Rel, which also contain transcription activation was examined after transient transfection with the ZAS3 expres- domains, may mediate ␬B transcription. However, significant sion construct by immunoblot analyses. In 38B9 cells, p65 was differences of these proteins were not detected in the preB mainly located in the cytoplasmic extracts and was barely cells (data not shown). Second, NF-␬B may have different detected from the nuclear extracts (Fig. 4, compare lanes 1 and posttranslational modifications. Recently, it is shown that 5). ZAS3 expression in 38B9 cells reduced the amount of nuclear phosphorylation and acetylation of p65 can increase NF-␬B p65 with a concomitant increase of cytoplasmic p65. On the transcriptional activity. Third, ZAS3 may compete for and contrary, p65 was located mainly in the nuclear extracts of displace NF-␬B from ␬B sites. ZAS3 may induce transcription Ϫ Ϫ ZAS3 / cells (Fig. 4, compare lanes 3 and 7). Introduction of less efficiently than NF-␬B or may even repress transcription. Ϫ Ϫ the ZAS3 expression construct in ZAS3 / cells resulted in a Members of the ZAS family have been shown to inhibit marked reduction of nuclear p65 and a corresponding increase transcription. ZAS1 negatively regulates the ␣1(II) collagen of cytoplasmic p65. As controls for protein loading and cell gene (27), whereas ZAS2 represses c- transcription from fractionation, appropriate signals were observed when immu- the major P2 promoter (28). noblot analyses were performed for a nuclear protein, histone To further elucidate the mechanisms by which ZAS3 regu- H1, or a cytosolic protein, heat-shock protein hsp90. We con- lates ␬B-mediated transcription, ␬B-reporter gene assays were clude that p65 was up-regulated in ZAS3Ϫ/Ϫ cells, and that ZAS3 performed with p65Ϫ/Ϫ MEF. The ␬B-reporter was 5-fold activated the nuclear export pathway of p65. more active in p65ϩ/ϩ than in p65Ϫ/Ϫ MEFs, demonstrating the A careful inspection revealed that the gel mobility of p65 from importance of p65 in ␬B-mediated transactivation (Fig. 5A). 38B9 cells was slightly faster than that of ZAS3Ϫ/Ϫ cells, indi- Notably, even when p65 was absent, ␬B-reporter gene activity cating differences in posttranslational modifications. Protein was still significantly higher than that of a control reporter, modifications at specific sites, including phosphorylation and which lacked the ␬B enhancer (data not shown). Therefore, the acetylation, have been shown to affect DNA binding, I␬B ␬B reporter must be driven by transacting factors for the

12304 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.2133048100 Hong et al. Downloaded by guest on September 25, 2021 Fig. 5. ZAS3 also represses transcription independent of p65. (A) Reporter gene assays. Transient transfection was performed in p65Ϫ/Ϫ MEFs (black bars) or p65ϩ/ϩ control MEFs (white bars). Normalized activity of the ␬B-reporter in p65ϩ/ϩ MEF transfected with the empty vector is taken as 100. A representative experiment of three is shown here. (B) ZASC inhibits transcription activation of VP16. Cos-7 cells were transiently transfected with pCMV110 (5 ␮g), and pSG-VP16, pSG-ZASC, or pSG424 expression plasmids, as indicated. The - tive chloramphenicol acetyl transferase activity of duplicates is illustrated for each experiment. The amounts of plasmid DNA used are : ϩ,10␮g; ϩϩ,15␮g; ϩϩϩ,20␮g; and Ϫ,0␮g. Activity from cells transfected with pCMV110 and Fig. 6. A model of ZAS3 in inhibiting NF-␬B. In the nucleus, ZAS3 interferes PSG-VP16 was assigned as 100. with NF-␬B-mediated transcription by competing for ␬B gene regulatory elements and by repressing transcription. In the cytoplasm, ZAS3, most likely a protein isoform, inhibits the nuclear translocation of p65 by association with ␬B-DNA in p65Ϫ/Ϫ MEFs. Finally, ZAS3 expression repressed TRAF2, which blocks the formation of the IKK complex. TRADD, TNF ␬ ϩ/ϩ Ϫ/Ϫ (TNFR)-associated ; RIP, receptor-interacting protein; TRAF2, B-reporter genes both in p65 and p65 MEF, suggesting ␬ that ZAS3 also represses ␬B-mediated transcription indepen- TNFR-associated factor 2; FADD, Fas-associated death domain; IKK, I B kinase; and NEMO, NF-␬B essential modulator. dent of p65. ZAS3-dependent transcription was studied by using a het- erologous system to differentiate DNA binding from transac- erally accelerated the growth of cell lines (32). In HeLa cells, tivation. Plasmid pSG-ZASC expressed the GAL4 DNA- ZAS3 deficiency led to anchorage-independent growth and the fused to the carboxyl one-third of the ZAS3 formation of multinucleated giant cells, in which nuclear division protein, including an acidic domain. Acidic domains are occurred without cell divisions. Gene knockout experiments commonly found in transcription factors and may interact with further show that ZAS2 and -3 are required for thymocyte the basal transcription machinery, transactivators, or adaptor maturation (15, 33). Several developmental anomalies, including molecules (29). Another plasmid, pSG-VP16, was similar to tumorigenesis, polydactyly, and hydronephrosis, were observed pSG-ZASC, except that the ZAS3 sequences were replaced Ϫ Ϫ Ϫ Ϫ in ZAS3 / RAG2 / chimeric mice (15). In addition, those

with the transactivation domain of VP16. VP16 is a strong ϩ ϩ IMMUNOLOGY mice showed a progressive depletion of CD4 CD8 double- transcriptional in mammalian cells (30). In this study, expression vectors were cotransfected with a reporter plasmid positive thymocytes, suggesting ZAS3 is important for the survival of T lymphocytes. Similarly, ZAS2 knockout mice were pCMV110, which contains four copies of GAL4-binding sites ϩ ϩ upstream of the bacterial chloramphenicol acetyl transferase severely depleted of CD4 and CD8 single-positive cells, a (CAT) gene, into a monkey kidney cell line, Cos-7. Expression defect contributed to failed positive selection (33). In addition, Ϸ ZAS proteins have been implicated in signal transduction. Shn of the CAT reporter was induced 50-fold by pSG-VP16 (Fig. ͞ ͞ 5B). Cotransfection with pSG-ZASC repressed the pSG-VP16- associates with Mad Smad to modulate decapentaplegic ␤ mediated transactivation of pCMV110. As a control, the transforming growth factor signaling during embryonic devel- ͞ parental vector, pSG424, containing the GAL4 DNA-binding opment (7). ZAS3 associates with TRAF2 to repress the TNF ␬ domain alone did not affect transactivation of pCMV110 by NF- B signal pathway (8). Furthermore, changes in gene pSG-VP16. The data show that ZAS3 inhibits transactivation expression of ZAS genes have been associated with poor prog- of VP16 and suggest that ZAS3 may function as a transcrip- nosis in chronic lymphocytic leukemia patients (34). We spec- tional repressor. ulate that ZAS3 may have a tumor suppression function. Previous studies have implicated ZAS in maintaining normal Here, we have shown that the subcellular localization of p65 growth. In Drosophila, expression of shn in cyst cells restricts and hence the major transactivation activity of NF-␬B are proliferation and mitosis of neighboring germ cells (31). Simi- modulated by ZAS3. A model for ZAS3 to regulate cellular larly, down-regulation of ZAS3 by an antisense construct gen- homeostasis is shown in Fig. 6. We propose that, whereas

Hong et al. PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12305 Downloaded by guest on September 25, 2021 NF-␬B activates transcription, ZAS3 mostly represses tran- governors of NF-␬B-mediated cell functions including apo- scription. In unstimulated cells, ZAS3 is responsible, at least ptosis, proliferation, inflammation, and immunity. in part, for guarding against aberrant NF-␬B-mediated trans- ϫ␬ ␬ activation and does so judiciously via multiple checkpoints: by We thank Dr. D. Guttridge for plasmids 3 B-Luc, p50 and p65, NF- B Ϫ/Ϫ ϩ/ϩ inhibiting the nuclear localization of p65, by competing for antibodies, and p65 and p65 MEFs; Dr. T. Hai (Ohio State Univer- ␬ sity) for HEK 293 cells; Dr. E. Clark (University of Washington, Seattle) for cis-acting B gene regulatory elements, and by serving as a p7ϫ␬B; and Dr. M. Ptashne (Memorial Sloan–Kettering Cancer Center, transcriptional repressor. In conclusion, this study establishes New York) for pSG424 and pCMV110. This work was supported in part by the groundwork for the ZAS family of zinc-finger proteins as a grant from the American Cancer Society, Ohio Division.

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