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B by ZAS3, a Zinc-Finger Protein That Also Binds to the B Motif

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B by ZAS3, a Zinc-Finger Protein That Also Binds to the B Motif Inhibition of NF-␬B by ZAS3, a zinc-finger protein 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 proteins are large zinc-finger transcriptional proteins (9, 10). This observation has prompted several groups, including implicated in growth, signal transduction, 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 gene 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 genes 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 transcription 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 lipid 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).
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