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The Journal of Immunology, 2002, 168: 680-688.
Copyright © 2002 by The American Association of Immunologists

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

Division of Infection, Immunity, Injury, and Repair, The Hospital for Sick Children, Toronto, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 (Krüppel-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 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acomplex series of cellular programs controls entry and exit from cell cycling in activated lymphocytes. Disruptions of critical checkpoints in these processes through genomic accidents in cell cycle control loci represent a key oncogenic event. In this study, we have used EBV as a model to analyze the transition of lymphocyte activation to growth transformation and identify a new zinc finger gene, ZNFEB, as a critical, cellular target molecule exploited by the virus for the induction of B cell growth transformation.

EBV, the human herpesvirus 4, infects the majority of humans and establishes latency in a small subset of the B lymphocyte compartment (1). In vitro, the virus activates B cells, triggering a classical, calcium-dependent activation cascade that is prerequisite for, but by itself insufficient for, subsequent growth transformation (2). Long-term growth transformation of the cells is associated with the coordinate expression of a selected repertoire of EBV-encoded genes, collectively known as the latency genes. Similar processes occur in vivo during acute infectious mononucleosis (3) and in healthy virus carriers (4), and they almost certainly play a role in the fulminant EBV-positive (EBV+) lymphomas of immunocompromised patients (5) or susceptible nonhuman primates (6, 7).

Thus, the analysis of differentially expressed genes in EBV-infected B cells has the dual goal to elucidate cellular signaling 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 and how they diverge toward oncogenic growth transformation. To this end, subtractive hybridization has been employed to delineate genes induced in primary B lymphocytes (8) or EBV-negative (EBV-) Burkitt’s lymphoma (BL)3 cell lines freshly infected with EBV. Several novel cellular genes associated with EBV-dependent growth transformation have been identified using this approach (9, 10, 11, 12).

We used cDNA subtraction cloning in the discovery of ZNFEB. It is a new element in B lymphocyte activation responses that EBV brings under viral control. Zinc finger proteins are usually DNA-binding transcription control proteins, and many function in cellular development and differentiation pathways. Members of this large protein family assume critical roles in cell cycle control, and their abnormal expression has been associated with the oncogenic transformation of host cells in which the protein resides. We believe that ZNFEB may serve a similar function in the EBV-driven growth transformation of B lymphocytes. Interestingly, the genomic localization of ZNFEB at 18q12 maps to a region in which chromosomal aberrations have been found in numerous different malignancies, including many of the B lineage (Mitelman Database of Chromosome Aberrations in Cancer, http://cgap.nci.nih.gov/Chromosomes/Mitelman), suggesting that ZNFEB is a candidate molecule involved in an even broader range of tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Subtractive hybridization and sequence analysis

Except for the use of the Uni-ZAP XR vector (pBluescript phagemid; Stratagene, La Jolla, CA), the preparation of a subtraction library containing cDNAs differentially expressed 6 h after EBV infection of normal tonsillar B cells in vitro was done as previously described (8). Briefly, purified mRNA of infected B cells was depleted of housekeeping transcripts through several cycles of hybridization to 20-fold excess of solid-phase cDNAs from noninfected B cells of the same tonsil. The remaining transcripts were cloned and plated at low density, and the inserts of randomly picked clones were directly amplified by PCR and sequenced on an automated DNA sequencer (Amersham Pharmacia Biotech, Mississauga, Ontario, Canada).

Cloning and chromosomal localization of ZNFEB

In addition to the initial 257-bp ZNFEB cDNA clone isolated from the subtraction library, seven overlapping cDNA clones were used to deduce the complete open reading frame (ORF) of ZNFEB. Two expressed sequence tags (ESTs) were obtained from the American Type Culture Collection (Manassas, VA). EST1 (I.M.A.G.E. CloneID 648205) (13) was isolated from a human teratocarcinoma cDNA library (Stratagene), and EST2 (ATCC 157317) was isolated from a Jurkat T cell cDNA library. Additional clones were identified in a Jurkat T cell cDNA library (Stratagene) and a human thymus {lambda}gt11 cDNA library (gifts of E. Arpaia, The Hospital for Sick Children, Toronto, Ontario, Canada). The chromosomal localization of ZNFEB employed the basic local alignment search tool algorithm (14) to search the National Center for Biotechnology Information Human Genome Sequence (15) and Celera Public Genome databases (16).

Cell culture and EBV infection

Mononuclear cells were purified from tonsils by Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) density centrifugation, and B lymphocytes were enriched (>=95%) by rosette depletion on a second Ficoll-Hypaque gradient. Cells were cultured in RPMI 1640 medium (Ontario Cancer Institute, Toronto, Ontario, Canada) supplemented with 10% heat-inactivated bovine calf serum (HyClone Sterile Systems, Logan, UT), L-glutamine (2 mM), penicillin, and streptomycin (50 µg/ml each; Life Technologies, Mississauga, Ontario, Canada). HepG2 and 293 cell lines were cultured in DMEM (Life Technologies) supplemented as above.

B95-8 is a semipermissive lymphoblastoid cell line (LCL) and provided all EBV employed in this study (see below) (17). The 2D5 is an EBV+ LCL established in our laboratory. Raji, Daudi, and Akata (18) are EBV+ BL lines. Akata- is an EBV- cell line derived from Akata (gift of K. Takada, Hokkaido University, Sapporo, Japan) (19). Ramos is an EBV- BL line. RI46 is an EBV- B cell line established in our laboratory from human PBMCs. Jurkat and CEM are human T cell leukemia lines. K562 (gift of N. Berinstein, University of Toronto, Toronto, Ontario, Canada), U937, and HL60 (gifts of F. Tsui, University of Toronto) are human myeloid leukemia cell lines with erythroid, monocytic, and myeloblastic features, respectively. HeLa (gift of N. Berinstein) is a human uterine cervical carcinoma cell line. HepG2 is a human hepatocellular carcinoma cell line, and 293 is an adenovirus E1-transformed human embryonic kidney cell line.

Plasmid p509 carries the genomic sequence of the immediate early EBV lytic gene BZLF1 under the control of the CMV promoter (gift of B. Sugden, University of Wisconsin Medical School, Madison, WI) (20, 21). B95-8 cells were transfected with p509 (10 µg/106 cells) and cultured for 7 days in the presence of n-butyrate (3 mM; Sigma-Aldrich). Supernatants were filtered (0.45-µm pore size), concentrated 4-fold by ultracentrifugation (210,000 x g for 2 h at 10°C), and frozen at -80°C in complete RPMI 1640 media. In all experiments, 1 ml of concentrated EBV/106 B cells were used for infection. Where indicated, EGTA was added to fresh B cells (5 mM) 1 h before virus infection. Total cellular RNA was isolated at the indicated times postinfection, as described below.

B cell activation assay

Freshly isolated tonsillar B cells (4 x 106/ml) were incubated with either rabbit anti-human IgM F(ab')2 (10 µg/ml; DAKO, Mississauga, Ontario, Canada) or a combination of PMA (10-8 M; Sigma-Aldrich) and ionomycin (0.5 µM; Sigma-Aldrich). Total cellular RNA was isolated at the indicated times posttreatment, as described below.

Nucleic acid isolation and analysis

Genomic DNA was isolated by standard protocols. Cellular RNA, purified using TRIzol Reagent as instructed (Life Technologies), was pretreated with ~1 U DNase I (Life Technologies) and reverse transcribed using 1 µg of poly(dT)12–18, ~80 U of RNAguard (Amersham Pharmacia Biotech), and 400 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies), as recommended by the manufacturer. cDNAs from human thymocytes, thymus, thyroid, exocrine pancreas, purified pancreatic islet cells, bone marrow, brain, lung, and skeletal muscle were gifts of W. Karges (Ulm University, Ulm, Germany) (22).

All RT-PCR reactions were conducted using the rTth DNA Polymerase GeneAmp XL PCR kit (PerkinElmer, Mississauga, Ontario, Canada) with the following primer pairs: ZNFEB (41, 5'-CGGTGACCAAGTCGAGATGG-3'; or 67, 5'-CAAGACCAAGATTGGAAAGCC-3'; and 2041A, 5'-CCAGTGCGGTTTTAATGTAGC-3'); ZNFEB splice variant (ZNFEBsv 57, 5'-GATTTTTCTGGTGGTCTAGTC-3' and 2041A); EBV nuclear Ag (EBNA)1 (109151, 5'-GTAGAAGGCCATTTTTCCAC-3'; and 109435A, 5'-TTTCTACGTGACTCCTAGCC-3'); {beta}-glucuronidase (GUS 1355, 5'-GTGATGTGGTCTGTGGCCAA-3'; and 1657A, 5'-TCTGCTCCATACTCGCTCTG-3'); {beta}-actin (actin 2445, 5'-ACTCTTCCAGCCTTCCTTCC-3'; and 3011A, 5'-TCATAGTCCGCCTAGAAGCA-3').

The ZNFEB 67 sequence is present in both ZNFEB and ZNFEBsv. The ZNFEB 41 primer is specific for sequences that flank the extra exon in ZNFEBsv, while the ZNFEBsv 57 primer is specific for sequences within this extra exon, and thus each will differentiate between splice variants as well as genomic DNA. RT-PCR products were electrophoretically separated. Where indicated, gels were blotted to Hybond-N+ membranes (Amersham Pharmacia Biotech) and hybridized with the following {gamma}-32P-end-labeled reporter probes (23, 24): ZNFEB and ZNFEBsv (ZNFEB 1858, 5'-CCATTGTGGAGAAGACAGTC-3'); EBNA1 (109194, 5'-TGAATACCACCAAGAAGGTG-3'); and {beta}-glucuronidase (GUS 1405, 5'-CTACTACTTGAAGATGGTGATCG-3'). Photography of gels/blots was performed using a Chemi Imager (Alpha Innotech, San Leandro, CA).

Recombinant protein production and 65Zn(II) blotting

DNA encoding the C-terminal (zinc finger) portion of ZNFEB (residues 252–423) was PCR amplified and cloned into the KpnI/XbaI sites of the GST fusion expression vector pGEX-4T-2 (GST-Cterm; Amersham Pharmacia Biotech). All constructs were sequence confirmed. GST-Cterm or pGEX-4T-2 vector alone was expressed in Escherichia coli BL21-CodonPlus(DE3)-RIL (Stratagene) at midlog growth phase by induction with 1 mM isopropyl {beta}-D-thiogalactoside for 2 h. Soluble protein was extracted (B-PER; Pierce, Rockford, IL) and added to glutathione Sepharose 4B (Amersham Pharmacia Biotech), washed, and eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.

65Zn(II) blotting was performed essentially as described (25), with the following modifications. Eluted protein, GST-Cterm, or GST alone (0.4 µg each) was separated by SDS-PAGE and blotted onto nitrocellulose in 10 mM 3-cyclohexylamino 1-propanesulfonic acid, pH 11 (Sigma-Aldrich). Duplicate samples were stained in a 0.25% (w/v) solution of Coomassie brilliant blue R250. Blotted membrane strips were equilibrated in three changes (20 min each) of 1x metal-binding buffer (100 mM Tris-HCl (pH 7), 50 mM NaCl, 1 mM DTT), and probed with ~1 µCi 65ZnCl2 (2 µM final Zn(II) concentration; NEN Life Science Products, Boston, MA) in 3 ml of 1x binding buffer without DTT for 1 h. Strips were washed twice in the same buffer and exposed to Kodak Biomax MR film (Kodak, Rochester, NY) for ~10 days at -80°C with intensifying screens.

Growth inhibition by antisense oligodeoxynucleotides

Unmodified sense and antisense oligodeoxynucleotides (10 µM) were added to triplicate cultures of purified tonsillar B cells (5 x 104/well) 1 h before EBV infection. In some experiments, a second dose of oligodeoxynucleotides of the same concentration was added 3 h (actin, p56lck, 5'/3' ZNFEB) or 16 h (BRLF1, BLLF3, BcLF1) after EBV infection. After 14 days, growth transformation was measured as [3H]thymidine uptake by liquid scintillation counting (2). Oligodeoxynucleotides used: BARF0 (160513, 5'-CCGCCAGAGTTCCAATAGAG-3'; 160533A, 5'-CTCTATTGGAACTCTGGCGG-3'); BARF1 (165571, 5'-CTTTCTTGGGTGAGCGAGTC-3'; 165590A, 5'-GACTCGCTCACCCAAGAAAG-3'); BLLF1 (gp350 92146, 5'-CTGACACACAAGCAAGGCTG-3'; 92127A, 5'-CTGACACACAAGCAAGGCTG-3'); EBNA1 (109401, 5'-TTTAAGAGCTCTCCTGGCTA-3'; 109420A, 5'-TAGCCAGGAGAGCTCTTAAA-3'); EBNA2 (48442, 5'-CAGGTACATGCCAACAACCT-3'; 48461A, 5'-AGGTTGTTGGCATGTACCTG-3'); BRLF1 (Rta 104900, 5'-GATGGAACATGCGTCGTTGC-3'; 104881A, 5'-GCAACGACGCATGTTCCATC-3'); BLLF3 (dUTPase 88460, ACACATACGCTACGCCTTCC-3'; 88441A, 5'-GGAAGGCGTAGCGTATGTGT-3'); BcLF1 (major capsid Ag 137041A, 5'-ATGCAGGATCTCCAGATCCA-3'); {beta}-actin (actin 846, 5'-CATGAAGTGTGACGTGGACA-3'; 1139A, 5'-TCATAGTCCGCCTAGAAGCA-3'); p56lck (lck 1223, 5'-CGCCAGAAGCCATTAACTA-3'; 1721A, 5'-GACAATGTGCAGAGTCCAC3'); 5' ZNFEB (67, 67A, 5'-GGCTTTCCAATCTTGGTCTTG-3'); 3' ZNFEB (1795, 5'-CCAGTGTGTTCAGTGCAGC-3', 2041A).

Nucleotide sequence accession numbers

The GenBank accession numbers for ZNFEB, HZF7 (26), the ZNFEBsv clones, ZNF-dp and PLACE1001304, ZNFphex133, and murine Zfp-35 (27, 28) are AF159567, X78930, AF153201, AK023456, AF373036, and X17617, respectively. The National Center for Biotechnology Information accession number and the Celera accession number for the chromosome 18 genomic contigs containing ZNFEB sequence are NT 010974 and Ga x2KMHMRU89G, respectively. Numbers designated for all oligodeoxynucleotides of EBV genes are based on the human herpesvirus 4, complete genome sequence (National Center for Biotechnology Information accession no. NC 001345).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Subtraction library screening

A subtraction library enriched for genes induced in human B cells 6 h after infection with EBV was previously made and successfully screened in our laboratory (8). In this study, we performed a second screening, randomly selecting and PCR-amplifying over 100 clones, of which 27 were fully sequenced. Four of the 27 had unknown gene sequences, including ZNFEB, which contained unique sequence as well as a consensus C2H2 (Krüppel-type) zinc finger motif. Since numerous zinc finger proteins have been implicated in cell differentiation and proliferation, we further characterized ZNFEB.

Cloning and sequence analysis of ZNFEB cDNAs

Sequence data obtained from the subtraction library, additional ESTs, and overlapping clones isolated from Jurkat T cell and human thymus cDNA libraries (see Materials and Methods) predict the ZNFEB mRNA to be at least 2195 bp long (Fig. 1Go). HZF7, a small clone of 199 bp (corresponding to bp 399–597 of the ZNFEB sequence), was previously isolated from the human monoblast cell line U937 (26). RT-PCRs performed on a human thymus cDNA library consistently gave two products when primers specific for the 5' untranslated region (UTR) of ZNFEB mRNA were used (Fig. 2GoA). We confirmed the existence of ZNFEBsv when two unpublished sequences in GenBank, ZNF-dp and PLACE1001304, were found to be identical to ZNFEB except for an extra 223 bp within this amplified 5' UTR region (Fig. 1Go). However, PCR analysis of the major ZNFEB ORF revealed that both reverse transcriptase and genomic amplicons were of identical size, suggesting that the coding portion of this gene is intronless (Fig. 2GoA). This lack of introns is not unprecedented among zinc finger genes (see Refs. 29, 30, 31).



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  		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 3' UTR sequence, including the poly(A) tail, found in the ZNFEBsv clone, ZNF-dp. The five zinc finger motifs are boxed.

 


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  		FIGURE 2. ZNFEB cDNA and genomic sequence analysis. Coding exons are boxed; introns are represented as dashed lines. The conceptual ORF is shown as a filled box. A, Splicing of ZNFEB RNA results in two variants (ZNFEB and ZNFEBsv). PCR analysis of a human thymus cDNA library (I, II) confirms that ZNFEBsv differs from ZNFEB by the addition of ~200 bp of sequence in the 5' UTR. The bulk of ZNFEB sequence is intronless, including the complete coding sequence (filled box) since a single, equivalent product is produced in both RT-PCR and genomic PCR amplifications (III). To rule out cDNA contamination, primers flanking an intron were used to amplify {beta}-actin. B, Physical map and positioning of the genomic locus of ZNFEB. The distances in kilobases between ZNFEB and its flanking genes, MAPRE2 and ZNF24, and their position on chromosome 18 are shown.

 
Identification of the ZNFEB genomic locus

Analysis of the ZNFEB genomic sequence in the National Center for Biotechnology Information and Celera public databases confirmed the intronless structure of the ZNFEB coding sequence and mapped the gene to chromosome 18q12 (Fig. 2GoB). The ZNFEB gene spans 18 kb of genomic sequence and is flanked by two known genes, the microtubule-associated protein RP/EB family member 2 (MAPRE2) (32, 33, 34) and another C2H2 zinc finger protein, ZNF24/KOX17 (35, 36) (Fig. 2GoB).

ZNFEB is a member of the C2H2 zinc finger protein family

The major ORF of ZNFEB and ZNFEBsv encodes a protein of 423 aa (Fig. 1Go) with a deduced mass of 48 kDa. The N-terminal portion of ZNFEB does not contain known sequence motifs. The C-terminal portion of the protein contains five putative zinc-binding finger domains (Fig. 1Go). These domains reveal a high conformity to the consensus CX2CX3FX5LX2HX3H sequence of the C2H2, or Krüppel-type, family of zinc finger proteins (Fig. 3GoA). Also highly conserved are the intervening 7 aa that separate each domain (the H/C-link) (37).



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  		FIGURE 3. Sequence comparison and zinc binding of the ZNFEB zinc finger domains. A, Alignment of the five ZNFEB zinc finger motifs and intervening amino acids (H/C-link) reveals high sequence conformity to the C2H2 (Krüppel-type) zinc finger family. The deduced consensus sequence (Css) is indicated at the bottom. Canonical C, F, L, and H residues are in bold. Other conserved residues are boxed. B, The C-terminal portion of ZNFEB (residues 252–423) containing all five zinc finger domains was purified from bacteria as a fusion to GST (GST-Cterm). Duplicate samples of 0.4 µg each of GST-Cterm or GST alone were separated by SDS-PAGE and stained (left side) or blotted onto nitrocellulose, and probed with ~1 µCi of 65Zn(II) (right side). Blots were exposed to Kodak Biomax MR film for ~10 days at -80°C with intensifying screens. GST-Cterm, but not GST, binds appreciable amounts of radiolabeled zinc.

 
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 of the ZNFEB C terminus, encompassing the 3' 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. 3GoB). 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 realized that ZNFEB possesses an unusual feature: 13 C2H2 zinc finger domains present in an alternate reading frame (one nucleotide shift; Fig. 4GoA). These domains are noncoding, due to the lack of an initiation codon and the presence of a stop codon within the 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. 4GoB). 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. 4GoB). Besides these minor changes, the ZNFphex133 cDNA sequence is identical to ZNFEBsv. In an alternate reading frame of ZNFphex133, the ZNFEB protein sequence is still encoded (Fig. 4GoB).



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  		FIGURE 4. Graphical representation of human and mouse ZNFEB homologs. A, ZNFEB has zinc finger domains in ORF (ZNFEBsv) and alternate 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 frameshift (see Discussion), is indicated (*). B, The human ZNFphex133 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 81–83% homology at the nucleotide level with the human genes, although protein sequences are widely divergent. D, Conserved intron-exon and splice site structures.

 
While ZNFEB and ZNFphex133 encode proteins with 5 and 13 zinc finger domains, respectively, a gene originally isolated from mouse testis, Zfp-35, retains the capacity to encode the complete set of 18 zinc finger domains (Fig. 4GoC) (27). Despite these extreme differences in protein, we believe that Zfp-35 is the murine homolog of ZNFEB, since the genomic intron/exon organization as well as splice junction sequences of both genes are virtually identical (Fig. 4GoD) (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 ~81 and ~83% contiguous with the corresponding ZNFEBsv and ZNFphex133 sequences, respectively, and ~78% 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. 5Go, A and B). Amplification of {beta}-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. 5GoA) and in primary human thymocytes (Fig. 5GoB). 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. 5GoA). ZNFEB transcripts are present in human thymus, thyroid, and pancreatic islet cells, but not in bone marrow, brain, lung, skeletal muscle, and exocrine pancreatic tissue (Fig. 5GoB).



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  		FIGURE 5. ZNFEB exhibits restricted expression in human cell lines and tissues. RT-PCR analysis was performed using cDNA prepared from a panel of human cell lines (A) and tissues (B). ZNFEB/ZNFEBsv expression is highest in the hemopoietic cell lines, Jurkat, CEM (T lymphocytic), and U937 (monocytic). Tissue expression includes thymocytes, thymus, and the nonlymphoid organs, thyroid, and pancreatic islets. H2O represents PCR amplification without template. Samples were normalized for {beta}-glucuronidase ({beta}-gluc.). RT-PCR products in A were Southern blotted and hybridized to radiolabeled internal reporter probes.

 
ZNFEB expression is transiently induced upon activation of B cells

Because ZNFEB was first isolated from a B lymphocyte cDNA 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. 6Go). We then measured ZNFEB expression in B cells stimulated with anti-IgM F(ab')2, or a phorbol ester plus calcium ionophore combination. As shown in Fig. 6Go, transient, high level expression of ZNFEB is seen 6 h after B cell Ag receptor stimulation, after which expression declines. Similarly, PMA/ionomycin-induced B cell activation results in the up-regulation of ZNFEB transcripts, albeit with faster kinetics; gene induction peaks at 2 h and returns to near basal levels by 12 h posttreatment. Thus, ZNFEB appears to be a new transiently induced B cell activation molecule.



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  		FIGURE 6. ZNFEB is transiently induced during B cell activation. Stimulation 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 {beta}-glucuronidase ({beta}-gluc.). Southern blotting of RT-PCR products was followed by hybridization with radiolabeled internal reporter probes.

 
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 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 6–12 h postinfection, but levels continued to rise, reaching high steady state levels over the ensuing 2 wk in culture (Fig. 7GoA). 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).



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  		FIGURE 7. ZNFEB is induced by EBV and is maintained in EBV+ B cell lines. A, Southern blotting of RT-PCR products, followed by hybridization with radiolabeled internal reporter probes, reveals an increase in expression of both ZNFEB and ZNFEBsv beginning at 6 h post-EBV infection of primary tonsillar B cells in vitro and continuing over a 2-wk culture period. RNA was purified from ~106 cells. Samples were normalized for {beta}-glucuronidase ({beta}-gluc.). B, Preincubation of tonsillar B cells with the calcium chelator, EGTA, does not affect the induction of ZNFEB/ZNFEBsv transcripts upon EBV infection, showing that the increased expression of this gene is not due to postreceptor-binding events only. The 2D5 is an EBV+ LCL. C, ZNFEB/ZNFEBsv transcripts remain at a high level of expression in EBV+, but not in EBV- B cell lines when RT-PCR products are assessed as in A. H2O represents PCR amplification without template.

 
We next determined whether ZNFEB expression was induced as part of early viral gene expression programs or the postreceptor-binding signaling cascade, as has been previously shown for the cellular protein tyrosine kinase, p56lck (40), and the heat shock proteins (hsp)70 and 90 (41). B cells were preincubated with the calcium chelator, EGTA, 1 h before virus infection. RT-PCR data showed that ZNFEB (and its splice variant) continue to be expressed at high levels despite the blockade of receptor binding-induced calcium mobilization and its downstream sequelae (Fig. 7GoB).

Comparison of ZNFEB expression in various B cell lines revealed that high level ZNFEB expression is sustained in EBV+ but not EBV- B cell lines (Fig. 7GoC). Akata-, the EBV- subclone of the EBV+ Akata parental line (19), shows reduced ZNFEB expression. These data demonstrate that EBV not only causes the induction of ZNFEB, but also maintains its expression in growth-transformed B cells.

ZNFEB functions in the EBV-dependent transformation process

The induced expression of ZNFEB by EBV implied that this cellular gene may play a direct role in the growth transformation program of the virus. To test this hypothesis, we targeted ZNFEB gene expression by antisense oligodeoxynucleotides. Sense and antisense oligodeoxynucleotides were chosen from unique (non-zinc finger) sequences in both the 5' and 3' regions of ZNFEB to help increase the probability of finding an active transcript target site (42). Sense or antisense oligodeoxynucleotides were added to freshly purified tonsillar B cells 1 h before, and, in some experiments, again at 3 or 16 h after EBV infection to ensure maximum cytosolic levels during the initial period of ZNFEB induction. Sense and antisense oligodeoxynucleotides from the EBV latency genes, BARF0, BARF1, EBNA1, and EBNA2; the lytic cycle genes, BLLF1 (gp350), BRLF1 (Rta), BLLF3 (dUTPase), and BcLF1 (major capsid Ag); and the cellular genes, p56lck and {beta}-actin, were employed as controls. Targeting of EBNA1, EBNA2, and p56lck by antisense oligodeoxynucleotides was previously demonstrated to abrogate growth transformation, while the high level expressed {beta}-actin protein is expected to be impervious to this form of antisense targeting (40, 43).

Two weeks after EBV infection, growth transformation was assessed by [3H]thymidine uptake (Fig. 8GoA) and Ig secretion (data not shown) with similar results. Data in Fig. 8GoA were pooled from several experiments and expressed as a percentage of EBV-infected control B cells (set at 100%). As expected (40, 43), antisense, but not sense oligodeoxynucleotides from EBNA1, EBNA2, and p56lck sequences inhibited EBV-induced growth transformation (Fig. 8GoA). In this study, we show a similar inhibition of growth transformation with ZNFEB antisense oligodeoxynucleotides. Antisense targeting of all the nontransforming EBV lytic cycle genes, or of {beta}-actin, showed no effect on the growth of infected cells (Fig. 8GoA). Antisense targeting of the latently expressed BARF0 and BARF1 gene products also showed no effect on EBV-mediated B cell transformation consistent with data gathered from rEBV carrying deletions in either of these genes (44, 45). In addition, microscopic analysis of cultures treated with antisense oligodeoxynucleotides against ZNFEB or other known transformation-prerequisite genes contained evidence of extensive cell death, and very few LCL could be established compared with control cultures (data not shown). These observations assign an essential role to ZNFEB in the EBV-mediated transformation process.



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  		FIGURE 8. ZNFEB antisense treatment effectively and specifically abrogates EBV-dependent B cell growth transformation. Triplicate cultures of tonsillar B cells (5 x 104/well) were loaded (10 µM/1 h) with sense or antisense oligodeoxynucleotides. EBV was then added, and in some experiments (see Materials and Methods) was followed by a second dose of oligodeoxynucleotides of the same concentration 3 or 16 h later. A, Cells were harvested after 2 wk of culture to assess growth transformation by [3H]thymidine uptake (1 µCi/16 h). Results are expressed as the percentage of cellular proliferation compared with the EBV-infected control cells (set at 100%). ND, Not done. B, RNA was purified from cells treated with ZNFEB sense and antisense oligodeoxynucleotides at the indicated times post-EBV infection for RT-PCR analysis of transcripts. EBNA1 is an EBV latency gene. Samples were normalized for {beta}-glucuronidase. Southern blots of RT-PCR products were hybridized with radiolabeled internal reporter probes.

 
To confirm the action of the antisense oligodeoxynucleotides, we analyzed ZNFEB gene expression in treated and control cells by RT-PCR (Fig. 8GoB). No transcripts of ZNFEB or ZNFEBsv remained 24 h after antisense treatment and EBV infection. Neither EBNA1 nor {beta}-glucuronidase expression was affected by ZNFEB antisense oligodeoxynucleotides, nor did any of the sense oligodeoxynucleotides have detectable effects. Collectively, these data led us to conclude that sustained ZNFEB expression may be a critical element in the transition of B cell activation to growth transformation by EBV, while transient expression is part of the normal lymphocyte activation program.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this work, we describe the discovery and characterization of a new C2H2 (Krüppel-type) zinc finger gene, ZNFEB, 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 accomplished, in large part, through the cloning of chromosomal junctions 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 fused with the fibroblast growth factor receptor 1 gene in the t(8;13) leukemia/lymphoma syndrome (50). The chromosomal localization of ZNFEB at 18q12 maps a hot spot for structural cytogenetic changes in numerous different malignancies (Mitelman Database of Chromosome Aberrations in Cancer, http://cgap. nci.nih.gov/Chromosomes/Mitelman). Predominant in the list of over 100 cases are myeloid and lymphoid leukemias, with acute B lineage lymphoblastic leukemia being the largest single class. Four separate cases in this group have translocations involving 18q12 with 12p11–13 (51, 52, 53). In line with the expression pattern of ZNFEB, there is the possibility that ZNFEB represents a new candidate oncogene that may be altered in these malignancies.

Flanking the ZNFEB 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 was shown to correlate with high proliferative states in multiple cell types (33). This report stated that B cells, like T cells, show an induction of MAPRE2. ZNF24 was one of 30 zinc finger genes isolated from human T cell lines. The presence of ZNF24 in close proximity to ZNFEB is consistent with the fact that genomic locations for zinc finger genes tend to cluster (54, 55, 56, 57).

Analysis of the cDNA and genomic sequences of ZNFEB reveals a lack of introns throughout most of the gene, including the complete coding sequence. Typically, within a C2H2 zinc finger protein, zinc finger domains are arranged in tandem, and are organized in a single exon in the genomic locus (58). Although it appears that the 13 zinc finger domains present in an alternate reading frame of ZNFEB are noncoding, it is tempting to speculate that at one point these domains may have been translated along with the 5' ORF zinc finger domains in the context of a larger protein. Arguments for this stem from the fact that the amino acid sequence connecting these two groups of zinc finger domains (noncoding and coding) represents a consensus, intrazinc finger H/C-link with the addition of only a single guanosine nucleotide responsible for the possible frameshift (see Fig. 4GoA). The 18 domains span most of the transcribed ZNFEB sequence, and their corresponding single exon could account for the intronless genomic structure of ZNFEB. Indeed, the mouse homolog is transcribed as a single, 18 zinc finger protein. If it is correct that the human sequence variants identified reflect derivatives evolved from an ancestral single gene resembling the 18 zinc finger murine gene, it would imply that the function of this ancestral gene was replaced by two new human genes with different DNA-binding regions and unrelated N- and C-terminal unique fragments.

Introns are present in the 5' UTR of ZNFEB and allow for differential splicing and production of a variant transcript containing an extra noncoding exon of 223 bp. Two unpublished cDNA sequences in GenBank, one isolated from human placenta (PLACE1001304) and the other from a human dermal hair papilla cell library (ZNF-dp), were of this variant. The reason for the production of these two mRNA species is not known. When tested, for instance in the induction by EBV infection in B cells (Fig. 7GoA), their expression appears to be virtually equivalent. ZNFEB transcript analysis using commercially available multiple tissue Northern blots was unsuccessful due to lack of sensitivity. Both variants code for the same protein, but regulation of translation may differ between the two. Unfortunately, ZNFEB protein expression in cells and tissues, like the majority of zinc finger-containing transcription factors (59), was at extremely low levels, precluding purification and analysis.

Although we were unable to isolate full-length protein, we could still gain an understanding of the function of ZNFEB through antisense oligodeoxynucleotide-mediated gene inhibition. Previous studies in our laboratory demonstrated the remarkable ability of human B cells to import oligodeoxynucleotides and the high degree of gene specificity of these unmodified molecules (8, 40, 43). The inhibition of ZNFEB expression during EBV infection of primary B cells shows striking effects on the subsequent growth transformation of the cells, similar to that seen with inhibition of genes known to participate in the growth transformation process, such as the viral EBNA1 and EBNA2 genes and the cellular tyrosine kinase, p56lck (40, 43). Importantly, all tested genes that are known not to play important roles in EBV-dependent B cell transformation were appropriately not affected by antisense targeting, including the lytic cycle and the nonessential latent genes, BARFO and BARF1 (44, 45).

There is currently no prescribed method for choosing the most effective antisense oligomers for any given gene, so selection of optimum target sites continues to be done on a trial-and-error basis (42). In our experiment, we would conclude that the 5' ZNFEB antisense oligodeoxynucleotide was superior over the 3' antisense reagent, as evidenced by its greater negative effect on EBV-induced proliferation.

The mechanism of action of antisense oligodeoxynucleotides involves RNase H-mediated degradation of transcripts carrying an RNA-DNA duplex, thus leading to specific gene inhibition (60). The RT-PCR analysis of cells incubated with ZNFEB antisense oligodeoxynucleotides confirmed the specific degradation of the ZNFEB transcripts and showed a complete disappearance of the mRNA by 24 h posttreatment (Fig. 8GoB). Essentially then, the effect of ZNFEB antisense treatment was the conversion of ZNFEB expression in these cells to a transient activation pattern, reminiscent of the profiles of ZNFEB expression in primary B cells stimulated by anti-IgM or PMA/ionomycin (Fig. 5Go). This is contrary to that seen in freshly EBV-infected cells and EBV+ BL lines. A steady rise in ZNFEB expression follows the primary EBV infection of B cells in vitro (Fig. 7GoA), and high level ZNFEB expression is maintained in EBV+ BL lines but not in EBV- B cell lines (Fig. 7GoB). Like the EBV+ cell lines, the primary B cells infected with EBV can live and multiply indefinitely. In this respect, the endurance of the ZNFEB signal correlates with the growth potential of the cells. This is not unlike malignancies that arise due to the deregulated expression of other zinc finger genes, as mentioned above.

Several lines of evidence make it tempting to speculate that ZNFEB may be a downstream effector of EBV latent protein(s) function. First, previous studies in our laboratory have shown that although calcium chelation effectively blocks transformation-prerequisite postreceptor-binding events, including the rapid induction of p56lck, hsp70, and hsp90, expression of the viral latent genes in such treated cells is unaffected (40). In this study, we show that ZNFEB induction is also resistant to EGTA treatment, suggesting that the alteration of its normal expression pattern in EBV-infected cells may be controlled by an EBV latency protein rather than as part of the postreceptor-binding signaling program. The kinetics of ZNFEB induction correlates well with that of the EBV latency genes (40). Finally, we have obtained preliminary evidence that ZNFEB may indeed be trans-activated by a critical, early EBV latency gene, latent membrane protein 1 (LMP1) (61, 62, 63). These experiments employed stable EBV-negative B cell transfectants with inducible LMP1 expression. We are in the process of characterizing the potential role of LMP1 in the induction of ZNFEB.

Collectively, we believe that by virtue of its sequence homology with other C2H2 (Krüppel-type) zinc finger family members and its ability to bind zinc, ZNFEB can transcriptionally activate (or repress) genes that are required for (or prevent) the transition of B lymphocyte activation to growth transformation. It will be important to delineate the molecular pathways that are activated by ZNFEB to understand why EBV uses this cellular gene for its own purposes, and for determining whether ZNFEB plays a broader role in cell cycle control. If the ZNFEB locus was found to be targeted in the lymphoma/leukemia-associated genomic accidents mapped to 18q12, this would be consistent with and strengthen this premise.


   Acknowledgments
 
We thank I. Miyazaki for the subtraction cDNA library and R. Cheung, A. Martin, M. Tsay, N. Sharfe, A. Freywald, and especially E. Arpaia for excellent advice and technical assistance. We also thank M. Scharff for critical reading of the manuscript.


   Footnotes
 
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. Back

2 Address correspondence and reprint requests to Dr. H.-Michael Dosch, Division of Infection, Immunity, Injury, and Repair, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, Canada M5G 1X8. Back

3 Abbreviations used in this paper: BL, Burkitt’s lymphoma; EBNA, EBV nuclear Ag; EST, expressed sequence tag; hsp, heat shock protein; LCL, lymphoblastoid cell line; LMP1, latent membrane protein 1; ORF, open reading frame; UTR, untranslated region; ZNFEBsv, ZNFEB splice variant. Back

Received for publication August 13, 2001. Accepted for publication November 12, 2001.


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J. Harper, L. Yan, R. M. Loureiro, I. Wu, J. Fang, P. A. D'Amore, and M. A. Moses
Repression of Vascular Endothelial Growth Factor Expression by the Zinc Finger Transcription Factor ZNF24
Cancer Res., September 15, 2007; 67(18): 8736 - 8741.
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