Transformation Activation to Oncogenic Growth Critical for The

Sustained Expression of the Novel EBV-Induced Zinc Finger Gene, ZNFEB, Is Critical for the Transition of B Lymphocyte Activation to Oncogenic Growth This information is current as Transformation of September 25, 2021. Cathryn E. Tune, Marc Pilon, Yuriko Saiki and H.-Michael Dosch J Immunol 2002; 168:680-688; ; doi: 10.4049/jimmunol.168.2.680 Downloaded from http://www.jimmunol.org/content/168/2/680

References This article cites 63 articles, 26 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/168/2/680.full#ref-list-1

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists by guest on September 25, 2021 • Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Sustained Expression of the Novel EBV-Induced Zinc Finger Gene, ZNFEB, Is Critical for the Transition of B Lymphocyte Activation to Oncogenic Growth Transformation1

Cathryn E. Tune, Marc Pilon, Yuriko Saiki, and H.-Michael Dosch2

EBV is a human tumor virus that infects and establishes latency in the majority of humans worldwide. In vitro, EBV growth transforms primary B lymphocytes into lymphoblastoid cell lines with high efficiency. We have used cDNA subtraction cloning to identify cellular target genes required for growth transformation and identified a new C2H2 (Kru¬ppel-type) zinc finger gene, ZNFEB, that is trans-activated early following EBV infection. In this study, we characterize ZNFEB, including its intronless locus, and human and mouse protein variants. The gene is transiently expressed during normal lymphocyte activation, and its expression

is sustained in EBV-positive but not EBV-negative B cell lines. There is limited expression in nonhemopoietic tissues. Its critical Downloaded from role in the growth transformation of B lineage cells is indicated by the abrogation of transformation with antisense strategies. ZNFEB maps to chromosome 18q12, a region with mutations in numerous, predominantly hemopoietic malignancies. The Journal of Immunology, 2002, 168: 680Ð688.

complex series of cellular programs controls entry and and how they diverge toward oncogenic growth transformation. To exit from cell cycling in activated lymphocytes. Disrup- this end, subtractive hybridization has been employed to delineate http://www.jimmunol.org/ tions of critical checkpoints in these processes through genes induced in primary B lymphocytes (8) or EBV-negative A Ϫ 3 genomic accidents in cell cycle control loci represent a key onco- (EBV ) Burkitt’s lymphoma (BL) cell lines freshly infected with genic event. In this study, we have used EBV as a model to analyze EBV. Several novel cellular genes associated with EBV-dependent the transition of lymphocyte activation to growth transformation growth transformation have been identified using this approach and identify a new zinc finger gene, ZNFEB, as a critical, cellular (9Ð12). target molecule exploited by the virus for the induction of B cell We used cDNA subtraction cloning in the discovery of ZNFEB. growth transformation. It is a new element in B lymphocyte activation responses that EBV EBV, the human herpesvirus 4, infects the majority of humans brings under viral control. Zinc finger proteins are usually DNA- and establishes latency in a small subset of the B lymphocyte com- binding transcription control proteins, and many function in cel- by guest on September 25, 2021 partment (1). In vitro, the virus activates B cells, triggering a clas- lular development and differentiation pathways. Members of this sical, calcium-dependent activation cascade that is prerequisite for, large protein family assume critical roles in cell cycle control, and but by itself insufficient for, subsequent growth transformation (2). their abnormal expression has been associated with the oncogenic Long-term growth transformation of the cells is associated with the transformation of host cells in which the protein resides. We be- coordinate expression of a selected repertoire of EBV-encoded lieve that ZNFEB may serve a similar function in the EBV-driven genes, collectively known as the latency genes. Similar processes growth transformation of B lymphocytes. Interestingly, the occur in vivo during acute infectious mononucleosis (3) and in genomic localization of ZNFEB at 18q12 maps to a region in which healthy virus carriers (4), and they almost certainly play a role in chromosomal aberrations have been found in numerous different the fulminant EBV-positive (EBVϩ) lymphomas of immunocom- malignancies, including many of the B lineage (Mitelman Data- promised patients (5) or susceptible nonhuman primates (6, 7). base of Chromosome Aberrations in Cancer, http://cgap.nci.nih. Thus, the analysis of differentially expressed genes in EBV- gov/Chromosomes/Mitelman), suggesting that ZNFEB is a candi- infected B cells has the dual goal to elucidate cellular signaling date molecule involved in an even broader range of tumors. pathways involved in physiological B cell activation as well as those involved in B cell oncogenesis. It is important to understand which aspects of these two pathways overlap, and at what point Materials and Methods Subtractive hybridization and sequence analysis Except for the use of the Uni-ZAP XR vector (pBluescript phagemid; Strat- Division of Infection, Immunity, Injury, and Repair, The Hospital for Sick Children, agene, La Jolla, CA), the preparation of a subtraction library containing Toronto, Ontario, Canada cDNAs differentially expressed 6 h after EBV infection of normal tonsillar B cells in vitro was done as previously described (8). Briefly, purified Received for publication August 13, 2001. Accepted for publication November 12, 2001. mRNA of infected B cells was depleted of housekeeping transcripts through several cycles of hybridization to 20-fold excess of solid-phase The costs of publication of this article were defrayed in part by the payment of page cDNAs from noninfected B cells of the same tonsil. The remaining tran- charges. This article must therefore be hereby marked advertisement in accordance scripts were cloned and plated at low density, and the inserts of randomly with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Canadian Institutes for Health Research. C.E.T. is the recipient of a Canadian Institutes for Health Research Doctoral Research Award. 2 Address correspondence and reprint requests to Dr. H.-Michael Dosch, Division of 3 Abbreviations used in this paper: BL, Burkitt’s lymphoma; EBNA, EBV nuclear Infection, Immunity, Injury, and Repair, The Hospital for Sick Children, 555 Uni- Ag; EST, expressed sequence tag; hsp, heat shock protein; LCL, lymphoblastoid cell versity Avenue, Toronto, Ontario, Canada M5G 1X8. E-mail address: hmdosch@ line; LMP1, latent membrane protein 1; ORF, open reading frame; UTR, untranslated sickkids.on.ca region; ZNFEBsv, ZNFEB splice variant.

picked clones were directly amplified by PCR and sequenced on an auto- 2041A, 5Ј-CCAGTGCGGTTTTAATGTAGC-3Ј); ZNFEB splice variant mated DNA sequencer (Amersham Pharmacia Biotech, Mississauga, On- (ZNFEBsv 57, 5Ј-GATTTTTCTGGTGGTCTAGTC-3Ј and 2041A); EBV tario, Canada). nuclear Ag (EBNA)1 (109151, 5Ј-GTAGAAGGCCATTTTTCCAC-3Ј; and 109435A, 5Ј-TTTCTACGTGACTCCTAGCC-3Ј); ␤-glucuronidase EB Cloning and chromosomal localization of ZNF (GUS 1355, 5Ј-GTGATGTGGTCTGTGGCCAA-3Ј; and 1657A, 5Ј-TCT GCTCCATACTCGCTCTG-3Ј); ␤-actin (actin 2445, 5Ј-ACTCTTCCAGC In addition to the initial 257-bp ZNFEB cDNA clone isolated from the CTTCCTTCC-3Ј; and 3011A, 5Ј-TCATAGTCCGCCTAGAAGCA-3Ј). subtraction library, seven overlapping cDNA clones were used to deduce The ZNFEB 67 sequence is present in both ZNFEB and ZNFEBsv. The the complete open reading frame (ORF) of ZNFEB. Two expressed se- ZNFEB 41 primer is specific for sequences that flank the extra exon in quence tags (ESTs) were obtained from the American Type Culture Col- ZNFEBsv, while the ZNFEBsv 57 primer is specific for sequences within lection (Manassas, VA). EST1 (I.M.A.G.E. CloneID 648205) (13) was this extra exon, and thus each will differentiate between splice variants as isolated from a human teratocarcinoma cDNA library (Stratagene), and well as genomic DNA. RT-PCR products were electrophoretically sepa- EST2 (ATCC 157317) was isolated from a Jurkat T cell cDNA library. ϩ rated. Where indicated, gels were blotted to Hybond-N membranes Additional clones were identified in a Jurkat T cell cDNA library (Strat- (Amersham Pharmacia Biotech) and hybridized with the following ␥-32P- agene) and a human thymus ␭gt11 cDNA library (gifts of E. Arpaia, The end-labeled reporter probes (23, 24): ZNFEB and ZNFEBsv (ZNFEB 1858, Hospital for Sick Children, Toronto, Ontario, Canada). The chromosomal 5Ј-CCATTGTGGAGAAGACAGTC-3Ј); EBNA1 (109194, 5Ј-TGAAT localization of ZNFEB employed the basic local alignment search tool al- ACCACCAAGAAGGTG-3Ј); and ␤-glucuronidase (GUS 1405, 5Ј- gorithm (14) to search the National Center for Biotechnology Information CTACTACTTGAAGATGGTGATCG-3Ј). Photography of gels/blots was Human Genome Sequence (15) and Celera Public Genome databases (16). performed using a Chemi Imager (Alpha Innotech, San Leandro, CA). Cell culture and EBV infection Recombinant protein production and 65Zn(II) blotting Mononuclear cells were purified from tonsils by Ficoll-Hypaque (Sigma- DNA encoding the C-terminal (zinc finger) portion of ZNFEB (residues Downloaded from Aldrich, St. Louis, MO) density centrifugation, and B lymphocytes were 252Ð423) was PCR amplified and cloned into the KpnI/XbaI sites of the Ն enriched ( 95%) by rosette depletion on a second Ficoll-Hypaque gradi- GST fusion expression vector pGEX-4T-2 (GST-Cterm; Amersham Phar- ent. Cells were cultured in RPMI 1640 medium (Ontario Cancer Institute, macia Biotech). All constructs were sequence confirmed. GST-Cterm or Toronto, Ontario, Canada) supplemented with 10% heat-inactivated bovine pGEX-4T-2 vector alone was expressed in Escherichia coli BL21-Codon- calf serum (HyClone Sterile Systems, Logan, UT), L-glutamine (2 mM), Plus(DE3)-RIL (Stratagene) at midlog growth phase by induction with 1 ␮ penicillin, and streptomycin (50 g/ml each; Life Technologies, Missis- mM isopropyl ␤-D-thiogalactoside for 2 h. Soluble protein was extracted sauga, Ontario, Canada). HepG2 and 293 cell lines were cultured in (B-PER; Pierce, Rockford, IL) and added to glutathione Sepharose 4B

DMEM (Life Technologies) supplemented as above. (Amersham Pharmacia Biotech), washed, and eluted with 10 mM reduced http://www.jimmunol.org/ B95-8 is a semipermissive lymphoblastoid cell line (LCL) and provided ϩ glutathione in 50 mM Tris-HCl, pH 8. all EBV employed in this study (see below) (17). The 2D5 is an EBV 65 ϩ Zn(II) blotting was performed essentially as described (25), with the LCL established in our laboratory. Raji, Daudi, and Akata (18) are EBV ␮ Ϫ Ϫ following modifications. Eluted protein, GST-Cterm, or GST alone (0.4 g BL lines. Akata is an EBV cell line derived from Akata (gift of K. each) was separated by SDS-PAGE and blotted onto nitrocellulose in 10 Takada, Hokkaido University, Sapporo, Japan) (19). Ramos is an EBVϪ Ϫ mM 3-cyclohexylamino 1-propanesulfonic acid, pH 11 (Sigma-Aldrich). BL line. RI46 is an EBV B cell line established in our laboratory from Duplicate samples were stained in a 0.25% (w/v) solution of Coomassie human PBMCs. Jurkat and CEM are human T cell leukemia lines. K562 brilliant blue R250. Blotted membrane strips were equilibrated in three (gift of N. Berinstein, University of Toronto, Toronto, Ontario, Canada), changes (20 min each) of 1ϫ metal-binding buffer (100 mM Tris-HCl (pH U937, and HL60 (gifts of F. Tsui, University of Toronto) are human my- ϳ ␮ 65 ␮ 7), 50 mM NaCl, 1 mM DTT), and probed with 1 Ci ZnCl2 (2 M eloid leukemia cell lines with erythroid, monocytic, and myeloblastic fea- final Zn(II) concentration; NEN Life Science Products, Boston, MA) in 3 tures, respectively. HeLa (gift of N. Berinstein) is a human uterine cervical ml of 1ϫ binding buffer without DTT for 1 h. Strips were washed twice in by guest on September 25, 2021 carcinoma cell line. HepG2 is a human hepatocellular carcinoma cell line, the same buffer and exposed to Kodak Biomax MR film (Kodak, Roches- and 293 is an adenovirus E1-transformed human embryonic kidney ter, NY) for ϳ10 days at Ϫ80¡C with intensifying screens. cell line. Plasmid p509 carries the genomic sequence of the immediate early EBV Growth inhibition by antisense oligodeoxynucleotides lytic gene BZLF1 under the control of the CMV promoter (gift of B. Sug- ␮ den, University of Wisconsin Medical School, Madison, WI) (20, 21). Unmodified sense and antisense oligodeoxynucleotides (10 M) were ϫ 4 B95-8 cells were transfected with p509 (10 ␮g/106 cells) and cultured for added to triplicate cultures of purified tonsillar B cells (5 10 /well) 1 h before EBV infection. In some experiments, a second dose of oligode- 7 days in the presence of n-butyrate (3 mM; Sigma-Aldrich). Supernatants lck were filtered (0.45-␮m pore size), concentrated 4-fold by ultracentrifuga- oxynucleotides of the same concentration was added 3 h (actin, p56 , ϫ Ϫ 5Ј/3Ј ZNFEB) or 16 h (BRLF1, BLLF3, BcLF1) after EBV infection. After tion (210,000 g for2hat10¡C), and frozen at 80¡C in complete RPMI 3 1640 media. In all experiments, 1 ml of concentrated EBV/106 B cells were 14 days, growth transformation was measured as [ H]thymidine uptake by used for infection. Where indicated, EGTA was added to fresh B cells (5 liquid scintillation counting (2). Oligodeoxynucleotides used: BARF0 Ј Ј Ј mM) 1 h before virus infection. Total cellular RNA was isolated at the (160513, 5 -CCGCCAGAGTTCCAATAGAG-3 ; 160533A, 5 -CTCTAT Ј Ј indicated times postinfection, as described below. TGGAACTCTGGCGG-3 ); BARF1 (165571, 5 -CTTTCTTGGGTGAG CGAGTC-3Ј; 165590A, 5Ј-GACTCGCTCACCCAAGAAAG-3Ј); BLLF1 B cell activation assay (gp350 92146, 5Ј-CTGACACACAAGCAAGGCTG-3Ј; 92127A, 5Ј-CT GACACACAAGCAAGGCTG-3Ј); EBNA1 (109401, 5Ј-TTTAAGAGCT Freshly isolated tonsillar B cells (4 ϫ 106/ml) were incubated with either CTCCTGGCTA-3Ј; 109420A, 5Ј-TAGCCAGGAGAGCTCTTAAA-3Ј); Ј ␮ rabbit anti-human IgM F(ab )2 (10 g/ml; DAKO, Mississauga, Ontario, EBNA2 (48442, 5Ј-CAGGTACATGCCAACAACCT-3Ј; 48461A, 5Ј-AGG Ϫ Canada) or a combination of PMA (10 8 M; Sigma-Aldrich) and ionomy- TTGTTGGCATGTACCTG-3Ј); BRLF1 (Rta 104900, 5Ј-GATGGAACA cin (0.5 ␮M; Sigma-Aldrich). Total cellular RNA was isolated at the in- TGCGTCGTTGC-3Ј; 104881A, 5Ј-GCAACGACGCATGTTCCATC-3Ј); dicated times posttreatment, as described below. BLLF3 (dUTPase 88460, ACACATACGCTACGCCTTCC-3Ј; 88441A, 5Ј-GGAAGGCGTAGCGTATGTGT-3Ј); BcLF1 (major capsid Ag 137041A, Nucleic acid isolation and analysis 5Ј-ATGCAGGATCTCCAGATCCA-3Ј); ␤-actin (actin 846, 5Ј-CATGAAG Ј Ј Ј Genomic DNA was isolated by standard protocols. Cellular RNA, purified TGTGACGTGGACA-3 ; 1139A, 5 -TCATAGTCCGCCTAGAAGCA-3 ); lck Ј Ј Ј using TRIzol Reagent as instructed (Life Technologies), was pretreated p56 (lck 1223, 5 -CGCCAGAAGCCATTAACTA-3 ; 1721A, 5 -GACA Ј Ј EB Ј with ϳ1 U DNase I (Life Technologies) and reverse transcribed using 1 ␮g ATGTGCAGAGTCCAC3 ); 5 ZNF (67, 67A, 5 -GGCTTTCCAA Ј Ј EB Ј of poly(dT) , ϳ80 U of RNAguard (Amersham Pharmacia Biotech), TCTTGGTCTTG-3 ); 3 ZNF (1795, 5 -CCAGTGTGTTCAGTG 12Ð18 Ј and 400 U of Moloney murine leukemia virus reverse transcriptase (Life CAGC-3 , 2041A). Technologies), as recommended by the manufacturer. cDNAs from human Nucleotide sequence accession numbers thymocytes, thymus, thyroid, exocrine pancreas, purified pancreatic islet cells, bone marrow, brain, lung, and skeletal muscle were gifts of W. The GenBank accession numbers for ZNFEB, HZF7 (26), the ZNFEBsv Karges (Ulm University, Ulm, Germany) (22). clones, ZNF-dp and PLACE1001304, ZNFphex133, and murine Zfp-35 All RT-PCR reactions were conducted using the rTth DNA Polymerase (27, 28) are AF159567, X78930, AF153201, AK023456, AF373036, and GeneAmp XL PCR kit (PerkinElmer, Mississauga, Ontario, Canada) with X17617, respectively. The National Center for Biotechnology Information the following primer pairs: ZNFEB (41, 5Ј-CGGTGACCAAGTC accession number and the Celera accession number for the chromosome 18 GAGATGG-3Ј; or 67, 5Ј-CAAGACCAAGATTGGAAAGCC-3Ј; and genomic contigs containing ZNFEB sequence are NT 010974 and 682 ZNFEB IS CRITICAL FOR EBV-INDUCED GROWTH TRANSFORMATION

Ga x2KMHMRU89G, respectively. Numbers designated for all oligode- plicated in cell differentiation and proliferation, we further char- oxynucleotides of EBV genes are based on the human herpesvirus 4, com- acterized ZNFEB. plete genome sequence (National Center for Biotechnology Information accession no. NC 001345).

EB Results Cloning and sequence analysis of ZNF cDNAs Subtraction library screening Sequence data obtained from the subtraction library, additional A subtraction library enriched for genes induced in human B cells ESTs, and overlapping clones isolated from Jurkat T cell and hu- 6 h after infection with EBV was previously made and successfully man thymus cDNA libraries (see Materials and Methods) predict EB screened in our laboratory (8). In this study, we performed a sec- the ZNF mRNA to be at least 2195 bp long (Fig. 1). HZF7, a EB ond screening, randomly selecting and PCR-amplifying over 100 small clone of 199 bp (corresponding to bp 399Ð597 of the ZNF clones, of which 27 were fully sequenced. Four of the 27 had sequence), was previously isolated from the human monoblast cell unknown gene sequences, including ZNFEB, which contained line U937 (26). RT-PCRs performed on a human thymus cDNA unique sequence as well as a consensus C2H2 (Kru¬ppel-type) zinc library consistently gave two products when primers specific for finger motif. Since numerous zinc finger proteins have been im- the 5Ј untranslated region (UTR) of ZNFEB mRNA were used (Fig. Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. ZNFEB cDNA and deduced protein sequence. The nucleotide and amino acid sequences are numbered on the right and left, respectively. The sequence and position of the extra 223-bp noncoding exon of the ZNFEBsv are shown. Underlined is the extra 3Ј UTR sequence, including the poly(A) tail, found in the ZNFEBsv clone, ZNF-dp. The ﬁve zinc ﬁnger motifs are boxed. The Journal of Immunology 683

2A). We confirmed the existence of ZNFEBsv when two unpub- gene spans 18 kb of genomic sequence and is flanked by two lished sequences in GenBank, ZNF-dp and PLACE1001304, were known genes, the microtubule-associated protein RP/EB family EB found to be identical to ZNF except for an extra 223 bp within member 2 (MAPRE2) (32Ð34) and another C2H2 zinc finger pro- this amplified 5Ј UTR region (Fig. 1). However, PCR analysis of tein, ZNF24/KOX17 (35, 36) (Fig. 2B). the major ZNFEB ORF revealed that both reverse transcriptase and EB genomic amplicons were of identical size, suggesting that the cod- ZNF is a member of the C2H2 zinc finger protein family ing portion of this gene is intronless (Fig. 2A). This lack of introns The major ORF of ZNFEB and ZNFEBsv encodes a protein of 423 is not unprecedented among zinc finger genes (see Refs. 29Ð31). aa (Fig. 1) with a deduced mass of 48 kDa. The N-terminal portion EB EB of ZNF does not contain known sequence motifs. The C-termi- Identification of the ZNF genomic locus nal portion of the protein contains five putative zinc-binding finger Analysis of the ZNFEB genomic sequence in the National Center domains (Fig. 1). These domains reveal a high conformity to the for Biotechnology Information and Celera public databases con- consensus CX2CX3FX5LX2HX3H sequence of the C2H2, or Kru¬p- firmed the intronless structure of the ZNFEB coding sequence and pel-type, family of zinc finger proteins (Fig. 3A). Also highly con- mapped the gene to chromosome 18q12 (Fig. 2B). The ZNFEB served are the intervening 7 aa that separate each domain (the H/C-link) (37). Transient mammalian, as well as bacterial and yeast expression of the complete coding sequence of ZNFEB, was unsuccessful, as was bacterial expression of the N-terminal (nonzinc finger-encoding) portion alone. However, we were able to measure expression Downloaded from of the ZNFEB C terminus, encompassing the 3Ј zinc finger motifs, as a GST fusion protein (GST-Cterm). After purification via glutathione affinity chromatography, we analyzed the zinc-binding capacity of these domains by zinc blotting and autoradiography (25). GST-Cterm was able to bind radioactive 65Zn(II), while GST alone

did not bind detectable amounts under the same conditions (Fig. http://www.jimmunol.org/ 3B). These data show that ZNFEB is a bona ﬁde zinc-binding protein and that the binding maps to the region containing the zinc ﬁnger domains.

Human and mouse ZNFEB protein variants Early on in our analysis of the complete cDNA sequence, we re- EB alized that ZNF possesses an unusual feature: 13 C2H2 zinc ﬁnger domains present in an alternate reading frame (one nucleo- by guest on September 25, 2021 tide shift; Fig. 4A). These domains are noncoding, due to the lack of an initiation codon and the presence of a stop codon within the

FIGURE 2. ZNFEB cDNA and genomic sequence analysis. Coding ex- FIGURE 3. Sequence comparison and zinc binding of the ZNFEB zinc ons are boxed; introns are represented as dashed lines. The conceptual ORF finger domains. A, Alignment of the five ZNFEB zinc finger motifs and is shown as a filled box. A, Splicing of ZNFEB RNA results in two variants intervening amino acids (H/C-link) reveals high sequence conformity to EB EB (ZNF and ZNF sv). PCR analysis of a human thymus cDNA library (I, the C2H2 (Kru¬ppel-type) zinc finger family. The deduced consensus se- II) confirms that ZNFEBsv differs from ZNFEB by the addition of ϳ200 bp quence (Css) is indicated at the bottom. Canonical C, F, L, and H residues of sequence in the 5Ј UTR. The bulk of ZNFEB sequence is intronless, are in bold. Other conserved residues are boxed. B, The C-terminal portion including the complete coding sequence (filled box) since a single, equiv- of ZNFEB (residues 252Ð423) containing all five zinc finger domains was alent product is produced in both RT-PCR and genomic PCR amplifica- purified from bacteria as a fusion to GST (GST-Cterm). Duplicate samples tions (III). To rule out cDNA contamination, primers flanking an intron of 0.4 ␮g each of GST-Cterm or GST alone were separated by SDS-PAGE were used to amplify ␤-actin. B, Physical map and positioning of the and stained (left side) or blotted onto nitrocellulose, and probed with ϳ1 genomic locus of ZNFEB. The distances in kilobases between ZNFEB and its ␮Ci of 65Zn(II) (right side). Blots were exposed to Kodak Biomax MR film flanking genes, MAPRE2 and ZNF24, and their position on chromosome 18 for ϳ10 days at Ϫ80¡C with intensifying screens. GST-Cterm, but not are shown. GST, binds appreciable amounts of radiolabeled zinc. 684 ZNFEB IS CRITICAL FOR EBV-INDUCED GROWTH TRANSFORMATION

tical (Fig. 4D) (28), and since Zfp-35 maps to mouse chromosome 18, syntenic to human chromosome 18. At the nucleotide level, the Zfp-35 coding sequence is ϳ81 and ϳ83% contiguous with the corresponding ZNFEBsv and ZNFphex133 sequences, respectively, and ϳ78% in the unique (nonzinc ﬁnger) region (data not shown).

ZNFEB expression in human cell lines and tissues RT-PCR data revealed a restricted expression pattern of ZNFEB in human cell lines and tissues (Fig. 5, A and B). Ampliﬁcation of ␤-glucuronidase served as a control since it is constitutively expressed at similar, but low, levels in most tissue (38). Highest expression of ZNFEB is seen in T lymphocytes, both in cell lines (Jurkat, CEM) (Fig. 5A) and in primary human thymocytes (Fig. 5B). Moderate expression is present in the monoblast cell line, U937, while other hemopoietic cell lines (K562, HL60), as well as nonhemopoietic cell lines (293, HeLa, HepG2), appear to express little, if any, ZNFEB (Fig. 5A). ZNFEB transcripts are present in human thymus, thyroid, and pancreatic islet cells, but not in bone Downloaded from marrow, brain, lung, skeletal muscle, and exocrine pancreatic tissue (Fig. 5B).

ZNFEB expression is transiently induced upon activation of B cells

Because ZNFEB was first isolated from a B lymphocyte cDNA http://www.jimmunol.org/ library, we next analyzed gene expression patterns in freshly isolated and in acutely activated tonsillar B cells. RT-PCR analysis of fresh tonsillar B cells demonstrated low ZNFEB expression levels (Fig. 6). We then measured ZNFEB expression in B cells stimu- Ј lated with anti-IgM F(ab )2, or a phorbol ester plus calcium iono- phore combination. As shown in Fig. 6, transient, high level expression of ZNFEB is seen 6 h after B cell Ag receptor stimulation, after which expression declines. Similarly, PMA/ionomycin-in- by guest on September 25, 2021 EB FIGURE 4. Graphical representation of human and mouse ZNFEB ho- duced B cell activation results in the up-regulation of ZNF tran- mologs. A, ZNFEB has zinc finger domains in ORF (ZNFEBsv) and alter- scripts, albeit with faster kinetics; gene induction peaks at2hand nate reading frames (ARF; ZNFARF), the latter without initiation codon (bars above horizontal line), but with a stop (bars below horizontal line) in the ninth domain. An additional guanosine, possibly responsible for the B, The human ZNFphex133 .(ء) frameshift (see Discussion), is indicated possesses ORFs for both ZNFEBsv and ZNFARF, deleting a stop at position 429 and transverting the stop in the ninth domain to Ser. C, The murine homolog (Zfp-35) includes 18 zinc finger domains and has 81Ð83% homology at the nucleotide level with the human genes, although protein sequences are widely divergent. D, Conserved intron-exon and splice site structures. ninth finger domain. However, a survey of ZNFEB-like cDNAs in GenBank brought our attention to a recently submitted, unpublished sequence, ZNFphex133, which, remarkably, does contain an ORF for these 13 zinc finger domains (Fig. 4B). This is made possible by both a 5-nt deletion that shifts the coding frame of ZNFphex133 to the alternate frame containing the 13 domains, and a transversion from a guanosine to a cytidine that changes the stop codon in the ninth finger domain to a serine residue (Fig. 4B). Besides these minor changes, the ZNFphex133 cDNA sequence is identical to ZNFEBsv. In an alternate reading frame of ZNFphex133, the ZNFEB EB protein sequence is still encoded (Fig. 4B). FIGURE 5. ZNF exhibits restricted expression in human cell lines While ZNFEB and ZNFphex133 encode proteins with 5 and 13 and tissues. RT-PCR analysis was performed using cDNA prepared from a panel of human cell lines (A) and tissues (B). ZNFEB/ZNFEBsv expression zinc finger domains, respectively, a gene originally isolated from is highest in the hemopoietic cell lines, Jurkat, CEM (T lymphocytic), and mouse testis, Zfp-35, retains the capacity to encode the complete U937 (monocytic). Tissue expression includes thymocytes, thymus, and set of 18 zinc finger domains (Fig. 4C) (27). Despite these extreme the nonlymphoid organs, thyroid, and pancreatic islets. H2O represents differences in protein, we believe that Zfp-35 is the murine ho- PCR amplification without template. Samples were normalized for ␤-glu- EB molog of ZNF , since the genomic intron/exon organization as curonidase (␤-gluc.). RT-PCR products in A were Southern blotted and well as splice junction sequences of both genes are virtually iden- hybridized to radiolabeled internal reporter probes. The Journal of Immunology 685

FIGURE 6. ZNFEB is transiently induced during B cell activation. Stim- Ј ulation of primary tonsillar B cells with either anti-IgM F(ab )2 or PMA/ ionomycin results in the induction of ZNFEB transcripts that reach highest expression levels by 6 and 2 h posttreatment, respectively, as shown by RT-PCR analysis. ZNFEB induction is transient since expression levels are diminished by 12 h posttreatment. H2O represents PCR ampliﬁcation without template. Samples were normalized for ␤-glucuronidase (␤-gluc.). Southern blotting of RT-PCR products was followed by hybridization with radiolabeled internal reporter probes. Downloaded from returns to near basal levels by 12 h posttreatment. Thus, ZNFEB appears to be a new transiently induced B cell activation molecule.

EBV induces prolonged expression of ZNFEB in B lymphocytes EBV infection causes morphological and phenotypical changes in

B cells that resemble those transiently induced in cells activated by http://www.jimmunol.org/ Ag (reviewed in Ref. 39). However, the ZNFEB response to EBV infection was distinctly different from responses to nontransforming activation signals observed above. Thus, progressively rising ZNFEB gene expression was consistently observed 6Ð12 h postinfection, but levels continued to rise, reaching high steady state levels over the ensuing 2 wk in culture (Fig. 7A). As shown in this study for the EBNA1 viral latency gene, the ZNFEB expression kinetics in EBV-infected primary B cells approximate those of viral latency genes (40). by guest on September 25, 2021 We next determined whether ZNFEB expression was induced as part of early viral gene expression programs or the postreceptor- ϩ binding signaling cascade, as has been previously shown for the FIGURE 7. ZNFEB is induced by EBV and is maintained in EBV B cellular protein tyrosine kinase, p56lck (40), and the heat shock cell lines. A, Southern blotting of RT-PCR products, followed by hybrid- proteins (hsp)70 and 90 (41). B cells were preincubated with the ization with radiolabeled internal reporter probes, reveals an increase in expression of both ZNFEB and ZNFEBsv beginning at 6 h post-EBV in- calcium chelator, EGTA, 1 h before virus infection. RT-PCR data EB fection of primary tonsillar B cells in vitro and continuing over a 2-wk showed that ZNF (and its splice variant) continue to be expressed culture period. RNA was puriﬁed from ϳ106 cells. Samples were normal- at high levels despite the blockade of receptor binding-induced cal- ized for ␤-glucuronidase (␤-gluc.). B, Preincubation of tonsillar B cells cium mobilization and its downstream sequelae (Fig. 7B). with the calcium chelator, EGTA, does not affect the induction of ZNFEB/ Comparison of ZNFEB expression in various B cell lines re- ZNFEBsv transcripts upon EBV infection, showing that the increased ex- ϩ vealed that high level ZNFEB expression is sustained in EBV but pression of this gene is not due to postreceptor-binding events only. The not EBVϪ B cell lines (Fig. 7C). AkataϪ, the EBVϪ subclone of 2D5 is an EBVϩ LCL. C, ZNFEB/ZNFEBsv transcripts remain at a high ϩ Ϫ the EBVϩ Akata parental line (19), shows reduced ZNFEB expres- level of expression in EBV , but not in EBV B cell lines when RT-PCR sion. These data demonstrate that EBV not only causes the induc- products are assessed as in A.H2O represents PCR ampliﬁcation without tion of ZNFEB, but also maintains its expression in growth-trans- template. formed B cells.

EB genes, BARF0, BARF1, EBNA1, and EBNA2; the lytic cycle ZNF functions in the EBV-dependent transformation process genes, BLLF1 (gp350), BRLF1 (Rta), BLLF3 (dUTPase), and The induced expression of ZNFEB by EBV implied that this cel- BcLF1 (major capsid Ag); and the cellular genes, p56lck and ␤-ac- lular gene may play a direct role in the growth transformation tin, were employed as controls. Targeting of EBNA1, EBNA2, and program of the virus. To test this hypothesis, we targeted ZNFEB p56lck by antisense oligodeoxynucleotides was previously demon- gene expression by antisense oligodeoxynucleotides. Sense and strated to abrogate growth transformation, while the high level antisense oligodeoxynucleotides were chosen from unique (non- expressed ␤-actin protein is expected to be impervious to this form zinc finger) sequences in both the 5Ј and 3Ј regions of ZNFEB to of antisense targeting (40, 43). help increase the probability of finding an active transcript target Two weeks after EBV infection, growth transformation was as- site (42). Sense or antisense oligodeoxynucleotides were added to sessed by [3H]thymidine uptake (Fig. 8A) and Ig secretion (data freshly purified tonsillar B cells 1 h before, and, in some experi- not shown) with similar results. Data in Fig. 8A were pooled from ments, again at 3 or 16 h after EBV infection to ensure maximum several experiments and expressed as a percentage of EBV-in- cytosolic levels during the initial period of ZNFEB induction. fected control B cells (set at 100%). As expected (40, 43), anti- Sense and antisense oligodeoxynucleotides from the EBV latency sense, but not sense oligodeoxynucleotides from EBNA1, EBNA2, 686 ZNFEB IS CRITICAL FOR EBV-INDUCED GROWTH TRANSFORMATION

by EBV, while transient expression is part of the normal lymphocyte activation program.

Discussion In this work, we describe the discovery and characterization of a EB new C2H2 (Kru¬ppel-type) zinc finger gene, ZNF , that is induced in and required for the growth transformation of human B lymphocytes by EBV. ZNFEB was isolated from a subtraction library enriched in genes induced by EBV after 6 h of infection of tonsillar B cells. Previous screening of this library revealed its potential to delineate important molecular events occurring early in the pathway toward cellular transformation, at a time when EBV latency genes are initially activated within the B cell (40). Indeed, the EBV homolog of human IL-10, viral IL-10, was isolated from this library, initiating studies that confirmed it as a critical element, not only during viral replication (46), but in the establishment of latency and growth transformation in primary B cells (8, 47). It is in the context of lymphoid malignancies that many zinc finger proteins have been identified and studied. This was accom- Downloaded from plished, in large part, through the cloning of chromosomal junc- tions of genomic translocations in different types of lymphomas.

For instance, BCL-5 and BCL-6 are both C2H2 zinc finger genes that were localized to clusters of breakpoints at 3q27 in non- Hodgkins B cell lymphomas (48, 49), and ZNF198 was found to be FIGURE 8. ZNFEB antisense treatment effectively and specifically ab- fused with the fibroblast growth factor receptor 1 gene in the t(8; http://www.jimmunol.org/ rogates EBV-dependent B cell growth transformation. Triplicate cultures of tonsillar B cells (5 ϫ 104/well) were loaded (10 ␮M/1 h) with sense or 13) leukemia/lymphoma syndrome (50). The chromosomal local- EB antisense oligodeoxynucleotides. EBV was then added, and in some ex- ization of ZNF at 18q12 maps a hot spot for structural cytoge- periments (see Materials and Methods) was followed by a second dose of netic changes in numerous different malignancies (Mitelman oligodeoxynucleotides of the same concentration 3 or 16 h later. A, Cells Database of Chromosome Aberrations in Cancer, http://cgap. were harvested after 2 wk of culture to assess growth transformation by nci.nih.gov/Chromosomes/Mitelman). Predominant in the list of [3H]thymidine uptake (1 ␮Ci/16 h). Results are expressed as the percentage over 100 cases are myeloid and lymphoid leukemias, with acute B of cellular proliferation compared with the EBV-infected control cells (set lineage lymphoblastic leukemia being the largest single class. Four at 100%). ND, Not done. B, RNA was purified from cells treated with EB separate cases in this group have translocations involving 18q12 ZNF sense and antisense oligodeoxynucleotides at the indicated times with 12p11Ð13 (51Ð53). In line with the expression pattern of by guest on September 25, 2021 post-EBV infection for RT-PCR analysis of transcripts. EBNA1 is an EBV ZNFEB, there is the possibility that ZNFEB represents a new can- latency gene. Samples were normalized for ␤-glucuronidase. Southern blots of RT-PCR products were hybridized with radiolabeled internal re- didate oncogene that may be altered in these malignancies. EB porter probes. Flanking the ZNF gene on 18q12 are MAPRE2 (RP1) and ZNF24 (KOX17). Both of these genes, like ZNFEB, are expressed in lymphocytes. MAPRE2 was cloned from differential display of CD3/CD28-activated vs nonactivated T cells, and its expression and p56lck sequences inhibited EBV-induced growth transforma- was shown to correlate with high proliferative states in multiple tion (Fig. 8A). In this study, we show a similar inhibition of growth cell types (33). This report stated that B cells, like T cells, show an transformation with ZNFEB antisense oligodeoxynucleotides. An- induction of MAPRE2. ZNF24 was one of 30 zinc finger genes tisense targeting of all the nontransforming EBV lytic cycle genes, isolated from human T cell lines. The presence of ZNF24 in close or of ␤-actin, showed no effect on the growth of infected cells (Fig. proximity to ZNFEB is consistent with the fact that genomic loca- 8A). Antisense targeting of the latently expressed BARF0 and tions for zinc finger genes tend to cluster (54Ð57). BARF1 gene products also showed no effect on EBV-mediated B Analysis of the cDNA and genomic sequences of ZNFEB reveals cell transformation consistent with data gathered from rEBV car- a lack of introns throughout most of the gene, including the com- rying deletions in either of these genes (44, 45). In addition, plete coding sequence. Typically, within a C2H2 zinc finger pro- microscopic analysis of cultures treated with antisense oligode- tein, zinc finger domains are arranged in tandem, and are organized oxynucleotides against ZNFEB or other known transformation- in a single exon in the genomic locus (58). Although it appears that prerequisite genes contained evidence of extensive cell death, and the 13 zinc finger domains present in an alternate reading frame of very few LCL could be established compared with control cultures ZNFEB are noncoding, it is tempting to speculate that at one point (data not shown). These observations assign an essential role to these domains may have been translated along with the 5Ј ORF ZNFEB in the EBV-mediated transformation process. zinc finger domains in the context of a larger protein. Arguments To confirm the action of the antisense oligodeoxynucleotides, for this stem from the fact that the amino acid sequence connecting we analyzed ZNFEB gene expression in treated and control cells by these two groups of zinc finger domains (noncoding and coding) RT-PCR (Fig. 8B). No transcripts of ZNFEB or ZNFEBsv remained represents a consensus, intrazinc finger H/C-link with the addition 24 h after antisense treatment and EBV infection. Neither EBNA1 of only a single guanosine nucleotide responsible for the possible nor ␤-glucuronidase expression was affected by ZNFEB antisense frameshift (see Fig. 4A). The 18 domains span most of the tran- oligodeoxynucleotides, nor did any of the sense oligodeoxynucle- scribed ZNFEB sequence, and their corresponding single exon otides have detectable effects. Collectively, these data led us to could account for the intronless genomic structure of ZNFEB.In- conclude that sustained ZNFEB expression may be a critical ele- deed, the mouse homolog is transcribed as a single, 18 zinc finger ment in the transition of B cell activation to growth transformation protein. If it is correct that the human sequence variants identified The Journal of Immunology 687 reflect derivatives evolved from an ancestral single gene resem- Several lines of evidence make it tempting to speculate that bling the 18 zinc finger murine gene, it would imply that the func- ZNFEB may be a downstream effector of EBV latent protein(s) tion of this ancestral gene was replaced by two new human genes function. First, previous studies in our laboratory have shown that with different DNA-binding regions and unrelated N- and C-ter- although calcium chelation effectively blocks transformation-pre- minal unique fragments. requisite postreceptor-binding events, including the rapid induc- Introns are present in the 5Ј UTR of ZNFEB and allow for dif- tion of p56lck, hsp70, and hsp90, expression of the viral latent ferential splicing and production of a variant transcript containing genes in such treated cells is unaffected (40). In this study, we an extra noncoding exon of 223 bp. Two unpublished cDNA se- show that ZNFEB induction is also resistant to EGTA treatment, quences in GenBank, one isolated from human placenta suggesting that the alteration of its normal expression pattern in (PLACE1001304) and the other from a human dermal hair papilla EBV-infected cells may be controlled by an EBV latency protein cell library (ZNF-dp), were of this variant. The reason for the rather than as part of the postreceptor-binding signaling program. production of these two mRNA species is not known. When tested, The kinetics of ZNFEB induction correlates well with that of the for instance in the induction by EBV infection in B cells (Fig. 7A), EBV latency genes (40). Finally, we have obtained preliminary their expression appears to be virtually equivalent. ZNFEB tran- evidence that ZNFEB may indeed be trans-activated by a critical, script analysis using commercially available multiple tissue North- early EBV latency gene, latent membrane protein 1 (LMP1) (61Ð ern blots was unsuccessful due to lack of sensitivity. Both variants 63). These experiments employed stable EBV-negative B cell code for the same protein, but regulation of translation may differ transfectants with inducible LMP1 expression. We are in the pro- between the two. Unfortunately, ZNFEB protein expression in cells cess of characterizing the potential role of LMP1 in the induction and tissues, like the majority of zinc finger-containing transcription of ZNFEB. Downloaded from factors (59), was at extremely low levels, precluding purification Collectively, we believe that by virtue of its sequence homology and analysis. with other C2H2 (Kru¬ppel-type) zinc finger family members and its Although we were unable to isolate full-length protein, we could ability to bind zinc, ZNFEB can transcriptionally activate (or re- still gain an understanding of the function of ZNFEB through an- press) genes that are required for (or prevent) the transition of B tisense oligodeoxynucleotide-mediated gene inhibition. Previous lymphocyte activation to growth transformation. It will be impor- studies in our laboratory demonstrated the remarkable ability of tant to delineate the molecular pathways that are activated by http://www.jimmunol.org/ human B cells to import oligodeoxynucleotides and the high de- ZNFEB to understand why EBV uses this cellular gene for its own gree of gene specificity of these unmodified molecules (8, 40, 43). purposes, and for determining whether ZNFEB plays a broader role The inhibition of ZNFEB expression during EBV infection of pri- in cell cycle control. If the ZNFEB locus was found to be targeted mary B cells shows striking effects on the subsequent growth trans- in the lymphoma/leukemia-associated genomic accidents mapped formation of the cells, similar to that seen with inhibition of genes to 18q12, this would be consistent with and strengthen this known to participate in the growth transformation process, such as premise. the viral EBNA1 and EBNA2 genes and the cellular tyrosine kinase, p56lck (40, 43). Importantly, all tested genes that are known Acknowledgments by guest on September 25, 2021 not to play important roles in EBV-dependent B cell transforma- We thank I. Miyazaki for the subtraction cDNA library and R. Cheung, tion were appropriately not affected by antisense targeting, includ- A. Martin, M. Tsay, N. Sharfe, A. Freywald, and especially E. Arpaia for ing the lytic cycle and the nonessential latent genes, BARFO and excellent advice and technical assistance. We also thank M. Scharff for BARF1 (44, 45). critical reading of the manuscript. There is currently no prescribed method for choosing the most effective antisense oligomers for any given gene, so selection of References optimum target sites continues to be done on a trial-and-error basis 1. Babcock, G. J., L. L. Decker, M. Volk, and D. A. Thorley-Lawson. 1998. EBV Ј EB persistence in memory B cells in vivo. Immunity 9:395. (42). In our experiment, we would conclude that the 5 ZNF 2. Dosch, H.-M., P. Lam, M. F. Hui, T. Hibi, and R. K. Cheung. 1990. EBV utilizes antisense oligodeoxynucleotide was superior over the 3Ј antisense a unique activation pathway for the transformation of human B cells. Int. Immu- reagent, as evidenced by its greater negative effect on EBV-in- nol. 2:833. 3. Tierney, R. J., N. Steven, L. S. Young, and A. B. Rickinson. 1994. Epstein-Barr duced proliferation. virus latency in blood mononuclear cells: analysis of viral gene transcription The mechanism of action of antisense oligodeoxynucleotides during primary infection and in the carrier state. J. Virol. 68:7374. involves RNase H-mediated degradation of transcripts carrying an 4. Joseph, A. M., G. J. Babcock, and D. A. Thorley-Lawson. 2000. Cells expressing the Epstein-Barr virus growth program are present in and restricted to the naive RNA-DNA duplex, thus leading to specific gene inhibition (60). B-cell subset of healthy tonsils. J. Virol. 74:9964. The RT-PCR analysis of cells incubated with ZNFEB antisense 5. McKnight, J. L., H. Cen, S. A. Riddler, M. C. Breinig, P. A. Williams, M. Ho, and P. S. Joseph. 1994. EBV gene expression, EBNA antibody responses and oligodeoxynucleotides confirmed the specific degradation of the ϩ EB EBV peripheral blood lymphocytes in post-transplant lymphoproliferative dis- ZNF transcripts and showed a complete disappearance of the ease. Leuk. Lymphoma 15:9. mRNA by 24 h posttreatment (Fig. 8B). Essentially then, the effect 6. Shope, T. C., D. Dechairo, and G. Miller. 1973. Malignant lymphoma in cotton- EB EB top marmosets after inoculation with Epstein-Barr virus. Proc. Natl. Acad. Sci. of ZNF antisense treatment was the conversion of ZNF ex- USA 70:2487. pression in these cells to a transient activation pattern, reminiscent 7. Feichtinger, H., S.-L. Li, E. Kaaya, P. Putkonen, K. Gru¬newald, K. Weyrer, D. of the profiles of ZNFEB expression in primary B cells stimulated Bo¬ttiger, I. Ernberg, A. Linde, G. Biberfeld, and P. Biberfeld. 1992. A monkey model for Epstein Barr virus-associated lymphomagenesis in human acquired by anti-IgM or PMA/ionomycin (Fig. 5). This is contrary to that immunodeficiency syndrome. J. Exp. Med. 176:281. ϩ seen in freshly EBV-infected cells and EBV BL lines. A steady 8. Miyazaki, I., R. K. Cheung, and H.-M. Dosch. 1993. Viral interleukin 10 is rise in ZNFEB expression follows the primary EBV infection of B critical for the induction of B cell growth transformation by Epstein-Barr virus. EB J. Exp. Med. 178:439. cells in vitro (Fig. 7A), and high level ZNF expression is main- 9. Birkenbach, M., K. Josefsen, R. Yalamanchili, G. Lenoir, and E. Kieff. 1993. tained in EBVϩ BL lines but not in EBVϪ B cell lines (Fig. 7B). Epstein-Barr virus-induced genes: first lymphocyte-specific G protein-coupled ϩ peptide receptors. J. Virol. 67:2209. Like the EBV cell lines, the primary B cells infected with EBV 10. Devergne, O., M. Hummel, H. Koeppen, B. M. Le, E. C. Nathanson, E. Kieff, and can live and multiply indefinitely. In this respect, the endurance of M. Birkenbach. 1996. A novel interleukin-12 p40-related protein induced by the ZNFEB signal correlates with the growth potential of the cells. latent Epstein-Barr virus infection in B lymphocytes. J. Virol. 70:1143. 11. Mosialos, G., M. Birkenbach, R. Yalamanchili, T. VanArsdale, C. Ware, and This is not unlike malignancies that arise due to the deregulated E. Kieff. 1995. The Epstein-Barr virus transforming protein LMP1 engages sig- expression of other zinc finger genes, as mentioned above. naling proteins for the tumor necrosis factor receptor family. Cell 80:389. 688 ZNFEB IS CRITICAL FOR EBV-INDUCED GROWTH TRANSFORMATION

12. Mosialos, G., S. Yamashiro, R. W. Baughman, P. Matsudaira, L. Vara, 39. Young, L. S., C. W. Dawson, and A. G. Eliopoulos. 2000. The expression and F. Matsumura, E. Kieff, and M. Birkenbach. 1994. Epstein-Barr virus infection function of Epstein-Barr virus encoded latent genes. Mol. Pathol. 53:238. induces expression in B lymphocytes of a novel gene encoding an evolutionarily 40. Cheung, R. K., I. Miyazaki, and H.-M. Dosch. 1993. Unexpected patterns of conserved 55-kilodalton actin-bundling protein. J. Virol. 68:7320. Epstein-Barr virus gene expression during early stages of B cell transformation. 13. Lennon, G. G., C. Auffray, M. Polymeropoulos, and M. B. Soares. 1996. The Int. Immunol. 5:707. I.M.A.G.E. Consortium: an integrated molecular analysis of genomes and their 41. Cheung, R. K., and H.-M. Dosch. 1993. The growth transformation of human B expression. Genomics 33:151. cells involves superinduction of hsp70 and hsp90. Virology 193:700. 14. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman. 1990. Basic 42. Lebedeva, I., and C. A. Stein. 2001. Antisense oligonucleotides: promise and local alignment search tool. J. Mol. Biol. 215:403. reality. Annu. Rev. Pharmacol. Toxicol. 41:403. 15. Lander, E. S., L. M. Linton, B. Birren, C. Nusbaum, M. C. Zody, J. Baldwin, 43. Cheung, R. K., and H.-M. Dosch. 1991. The tyrosine kinase lck is critically K. Devon, K. Dewar, M. Doyle, W. FitzHugh, et al. 2001. Initial sequencing and involved in the growth transformation of human B lymphocytes. J. Biol. Chem. analysis of the human genome. Nature 409:860. 266:8667. 16. Venter, J. C., M. D. Adams, E. W. Myers, P. W. Li, R. J. Mural, G. G. Sutton, H. O. Smith, M. Yandell, C. A. Evans, R. A. Holt, et al. 2001. The sequence of 44. Robertson, E. S., B. Tomkinson, and E. Kieff. 1994. An Epstein-Barr virus with the human genome. Science 291:1304. a 58-kilobase-pair deletion that includes BARF0 transforms B lymphocytes in 17. Miller, G., and M. Lipman. 1973. Release of infectious Epstein-Barr virus by vitro. J. Virol. 68:1449. transformed marmoset leukocytes. Proc. Natl. Acad. Sci. USA 70:190. 45. Cohen, J. I., and K. Lekstrom. 1999. Epstein-Barr virus BARF1 protein is dis- ␣ 18. Takada, K., K. Horinouchi, Y. Ono, T. Aya, T. Osato, M. Takahashi, and pensable for B-cell transformation and inhibits interferon secretion from mono- S. Hayasaka. 1991. An Epstein-Barr virus-producer line Akata: establishment of nuclear cells. J. Virol. 73:7627. the cell line and analysis of viral DNA. Virus Genes 5:147. 46. Touitou, R., C. Cochet, and I. Joab. 1996. Transcriptional analysis of the Epstein- 19. Shimizu, N., H. Yoshiyama, and K. Takada. 1996. Clonal propagation of Epstein- Barr virus interleukin-10 homologue during the lytic cycle. J. Gen. Virol. 77: Barr virus (EBV) recombinants in EBV-negative Akata cells. J. Virol. 70:7260. 1163. 20. Hammerschmidt, W., and B. Sugden. 1988. Identification and characterization of 47. Miyazaki, I., R. K. Cheung, and H.-M. Dosch. 1994. The role of vIL-10 and other oriLyt, a lytic origin of DNA replication of Epstein-Barr virus. Cell 55:427. EBV latency genes early during B cell growth transformation. In Seminars in 21. Kempkes, B., D. Pich, R. Zeidler, and W. Hammerschmidt. 1995. Immortaliza- Virology, Vol. 5. M. E. Oldstone, ed. Academic, London, p. 405. Downloaded from tion of human primary B lymphocytes in vitro with DNA. Proc. Natl. Acad. Sci. 48. Ye, B. H., F. Lista, F. Lo Coco, D. M. Knowles, K. Offit, R. S. K. Chaganti, and USA 92:5875. R. Dalla-Favera. 1993. Alterations of a zinc finger-encoding gene: BCL- 6 in 22. Karges, W., M. Pietropaolo, C. A. Ackerley, and H.-M. Dosch. 1996. Gene ex- diffuse large-cell lymphoma. Science 262:747. pression of islet cell antigen p69 in human, mouse, and rat. Diabetes 45:513. 49. Miki, T., N. Kawamata, S. Hirosawa, and N. Aoki. 1994. Gene involved in the 23. Feinmesser, R., I. Miyazaki, R. Cheung, J. L. Freeman, A. M. Noyek, and H.-M. 3q27 translocation associated with B-cell lymphoma, BCL5, encodes a Kruppel- Dosch. 1992. Diagnosis of nasopharyngeal carcinoma by DNA amplification of like zinc-finger protein. Blood 83:26. tissue obtained by fine-needle aspiration. N. Engl. J. Med. 326:17. 50. Xiao, S., S. R. Nalabolu, J. C. Aster, J. Ma, L. Abruzzo, E. S. Jaffe, R. Stone, 24. Cheung, R. K., M. F. Hui, H.-M. Dosch, and T. E. Ewart. 1993. Detection of viral S. M. Weissman, T. J. Hudson, and J. A. Fletcher. 1998. FGFR1 is fused with a sequences by internally calibrated gene amplification. BioTechniques 14:785. novel zinc-finger gene, ZNF198, in the t(8;13) leukemia/lymphoma syndrome. http://www.jimmunol.org/ 25. DiDonato, M., S. Narindrasorasak, J. R. Forbes, D. W. Cox, and B. Sarkar. 1997. Nat. Genet. 18:84. Expression, purification, and metal binding properties of the N-terminal domain 51. Amor, D. J., E. M. Algar, H. R. Slater, and P. J. Smith. 1998. High frequency of from the Wilson disease putative copper-transporting ATPase (ATP7B). J. Biol. t(12;21) in childhood acute lymphoblastic leukemia detected by RT-PCR. Pa- Chem. 272:33279. thology 30:381. 26. Åbrink, M., M. Aveskogh, and L. Hellman. 1995. Isolation of cDNA clones for 42 different Kruppel-related zinc finger proteins expressed in the human mono- 52. Uckun, F. M., K. J. Gajl-Peczalska, A. J. Provisor, and N. A. Heerema. 1989. blast cell line U-937. DNA Cell Biol. 14:125. Immunophenotype-karyotype associations in human acute lymphoblastic leuke- 27. Cunliffe, V., P. Koopman, A. McLaren, and J. Trowsdale. 1990. A mouse zinc mia. Blood 73:271. finger gene which is transiently expressed during spermatogenesis. EMBO J. 53. Ramakers-van Woerden, N. L., R. Pieters, A. H. Loonen, I. Hubeek, 9:197. E. van Drunen, H. B. Beverloo, R. M. Slater, J. Harbott, J. Seyfarth, 28. Cunliffe, V., S. Williams, and J. Trowsdale. 1990. Genomic analysis of a mouse E. R. van Wering, et al. 2000. TEL/AML1 gene fusion is related to in vitro drug

zinc finger gene, Zfp-35, that is up-regulated during spermatogenesis. Genomics sensitivity for L- asparaginase in childhood acute lymphoblastic leukemia. Blood by guest on September 25, 2021 8:331. 96:1094. 29. Relaix, F., X. Weng, G. Marazzi, E. Yang, N. Copeland, N. Jenkins, S. E. Spence, 54. Lichter, P., P. Bray, T. Ried, I. B. Dawid, and D. C. Ward. 1992. Clustering of and D. Sassoon. 1996. Pw1, a novel zinc finger gene implicated in the myogenic C2ÐH2 zinc finger motif sequences within telomeric and fragile site regions of and neuronal lineages. Dev. Biol. 177:383. human chromosomes. Genomics 13:999. 30. Jong, M. T. C., T. A. Gray, Y. G. Ji, C. C. Glenn, S. Saitoh, D. J. Driscoll, and 55. Huebner, K., T. Druck, C. M. Croce, and H. J. Thiesen. 1991. Twenty-seven R. D. Nicholls. 1999. A novel imprinted gene, encoding a RING zinc-finger nonoverlapping zinc finger cDNAs from human T cells map to nine different protein, and overlapping antisense transcript in the Prader-Willi syndrome critical chromosomes with apparent clustering. Am. J. Hum. Genet. 48:726. region. Hum. Mol. Genet. 8:783. 56. Bellefroid, E. J., J. C. Marine, T. Ried, P. J. Lecocq, M. Riviere, C. Amemiya, 31. Lans, M. S., Q. Li, J. Lu, W. S. Modi, and A. L. Notkins. 1994. Genomic orga- D. A. Poncelet, P. G. Coulie, P. de Jong, and C. Szpirer. 1993. Clustered organization, 5Ј upstream sequence, and chromosomal localization of an insulinoma- nization of homologous KRAB zinc-finger genes with enhanced expression in associated intronless gene, IA-1. J. Biol. Chem. 269:14170. human T lymphoid cells. EMBO J. 12:1363. 32. Su, L.-K., and L. Qi. 2001. Characterization of human MAPRE genes and their 57. Rousseau-Merck, M. F., A. Tunnacliffe, R. Berger, B. A. Ponder, and proteins. Genomics 71:142. H. J. Thiesen. 1992. A cluster of expressed zinc finger protein genes in the 33. Renner, C., J.-P. Pfitzenmeier, K. Gerlach, G. Held, S. Ohnesorge, U. Sahin, pericentromeric region of human chromosome 10. Genomics 13:845. S. Bauer, and M. Pfreundschuh. 1997. RP1, a new member of the adenomatous 58. Pieler, T., and E. Bellefroid. 1994. Perspectives on zinc finger protein function polyposis coli-binding EB1-like gene family, is differentially expressed in acti- and evolution: an update. Mol. Biol. Rep. 20:1. vated T cells. J. Immunol. 159:1279. 59. Falchuk, K. H. 1998. Zinc transcription factors in cellular differentiation and 34. Juwana, J.-P., P. Henderikx, A. Mischo, A. Wadle, N. Fadle, K. Gerlach, organogenesis. J. Trace Elem. Exp. Med. 11:89. J. W. Arends, H. Hoogenboom, M. Pfreundschuh, and C. Renner. 1999. EB/RP gene family encodes tubulin binding proteins. Int. J. Cancer 81:275. 60. Wagner, R. W. 1994. Gene inhibition using antisense oligodeoxynucleotides. 35. Thiesen, H. J. 1990. Multiple genes encoding zinc finger domains are expressed Nature 372:333. in human T cells. New Biol. 2:363. 61. Kaye, K. M., K. M. Izumi, and E. Kieff. 1993. Epstein-Barr virus latent mem- 36. Rousseau-Merck, M. F., K. Huebner, R. Berger, and H. J. Thiesen. 1991. Chro- brane protein 1 is essential for B-lymphocyte growth transformation. Proc. Natl. mosomal localization of two human zinc finger protein genes, ZNF24 (KOX17) Acad. Sci. USA 90:9150. and ZNF29 (KOX26), to 18q12 and 17p13Ðp12, respectively. Genomics 9:154. 62. Kaye, K. M., K. M. Izumi, G. Mosialos, and E. Kieff. 1995. The Epstein-Barr 37. Schuh, R., W. Aicher, U. Gaul, S. Cote, A. Preiss, D. Maier, E. Seifert, virus LMP1 cytoplasmic carboxy terminus is essential for B-lymphocyte trans- U. Nauber, C. Schroder, and R. Kemler. 1986. A conserved family of nuclear formation; fibroblast cocultivation complements a critical function within the proteins containing structural elements of the finger protein encoded by Kruppel, terminal 155 residues. J. Virol. 69:675. a Drosophila segmentation gene. Cell 47:1025. 63. Izumi, K. M., K. M. Kaye, and E. D. Kieff. 1997. The Epstein-Barr virus LMP1 38. Karges, W. J. P., R. Gaedigk, and H.-M. Dosch. 1994. Quantitative analysis of amino acid sequence that engages tumor necrosis factor receptor associated fac- gene expression in different tissues by template-calibrated RT-PCR and laser- tors is critical for primary B lymphocyte growth transformation. Proc. Natl. Acad. induced fluorescence. PCR Methods Appl. 4:154. Sci. USA 94:1447.